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Molecular characterization of two cotton geminiviruses.
Item Type text; Dissertation-Reproduction (electronic)
Authors Nadeem, Athar.
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MOLECULAR CHARACTERIZATION OF TWO COrrON GEMINIVIRUSES
by
Athar Nadeem
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF PLANT PATHOLOGY
III Partial Fulfillment of the Requirements
for the degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
The University of Arizona
1995
UMI Number: 9531069
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THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have
read the dissertation prepared by Athar Nadeem -----------------------------------------entitled MOLECULAR CHARACTERIZATION OF TWO COTTON GEMINIVIRUSES
and recommend that it be accepted as fulfilling the dissertation
requirement for the Degree of Doctor of Philosophy/Plant Pathology
3~i;~ D.(te J
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Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my
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STATEMENT BY AUllIOR
l11is dissertation has been submitted 'in partial fulfillment of requirements for an
advances degree at the University of Ari7.0na and is deposited in the University Lihr;uy to
be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowahle without special pennissioll.
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4
ACKNOWLEDGMENTS
In the name of Allah, the Compassionate, the Merciful. Praise is only for Allah, the
Lord of the Universe, the Master of the Day of Judgment Whoever is grateful, his
gratefulness is for his own good, and who ever is ungrateful, then Allah is indeed Self
Sufficient and Self-Praiseworthy. I am grateful to my Lord who created me, gave me
knowledge and to whom I have to return.
I would like to express my profound gratitude to my advisors Drs. Zhongguo
Xiong and Merritt R Nelson for their invaluable guidance, and indispensable assistance
during every phase of this study. I am also very grateful to Drs. M. E. Stanghellini, L. S.
Pierson In and Frank Katterman who were always willing to help and guide me
throughout my graduate studies. My acknowledgments are also due to friends and
families, whose names if I mention individually will require at least few pages, they not
provided me the love but helped me in any difficulty I faced during my stay at Tucson.
Thanks to all other faculty staff and students of the department of Plant Pathology for
their cooperation and guidance.
Special thanks to my government, Pakistan Agricultural Research Council, US
Agency for International Development for providing me the opportunity for this study.
Thanks are due to Dr. Saif Khalid and his colleagues, for providing me the culture
of cotton leaf curl virus and laboratory facilities for DNA extraction and help in organizing
my trip to cotton growing areas of Multan, for sample collection.
At last but not least, I am thankful to My wife, and Children Marium, Fatimah, and
Sidrah, who were always complaining for my late staying in the laboratory but remained
patient and source of joy during the phase of completion of this dissertation. May Allah
reward them.
Dedicated
to my country the Islamic Republic of Pakistan
and my parents, who always pray for my success in this life and in hereafter
5
6
Table of contents
LIST OF FIGURES .............................................................................................................................. 10
LIST OF TABLES ....................................................................•.......................................................... 11
ABSTRACT .•.•................•.............................••.........••.................................•......................................... 12
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW ..••.........................••........•.........•.......................... 13
1. GEMINIVIRUSES INFEC11NG COTION .•.••.•••...•......•.•••••..••...•••••••••••••••••.••....•...•.......•••••••••••.•....•.•••.•....•.. 13
2. BIOLOGY OF GEMINIVIRUSES ••••••....•••••••.....•••••••.•.......••••••••.•.•....••....••..•.••.••••••••••••••••....•.•.•.•••.......•••••• 14
2.1 Synlptol1Ultology ........................................ ................................................................................... 15
2.2 Morphology ................................................................................................................................. 16
2.3 Tral/smissiol/ .......................................... ...................................................................................... 16
2.4 Gemil/ivirus subgroups ................................................................................................................ 17
2.5 Evolutiol/ary re/atiol/ship . ........................................................................................................... 17
3. MOLECULAR BIOLOGY OF GEMINIVIRUSES •••••.•....••••••••••••.....••••••••••••••.....••...•...•••••.•••..•...•..•••.•.••..•....... 19
3.1 Gel/ome orgal/i=atiol/ ................................................................................................................... 19
3.1.1 Subgroup 1 ............................................................................................................................................ 19
3.1.2 Subgroup II: .......................................................................................................................................... 20
3.1.3 Subgroup III ......................................................................................................................................... 21
3.2 Subgel/omic DNA ...................................................................................... ................................... 22
3.3 Replicatiol/ .................................................................................................................................. 23
3.3.1 Site of replication ................................................................................................................................. 23
3.3.2 Replication mechanism ......................................................................................................................... 23
3.3.3 Complementary strand Synthesis .......................................................................................................... 24
3.3.4 Synthesis of virion strand ..................................................................................................................... 25
3.4 Gel/e expressiol/ ......................................................................... .................................................. 26
3.4.1 Transcription ........................................................................................................................................ 26
3.4.2 Regulations of gene expression ............................................................................................................. 27
3.5. FUI/ctiol/ of the viral gel/es ................................................................................... ...................... 29
3.5.1 Function of the AC llC 1 protein ........................................................................................................... 29
3.5.2 Function of AC2 protein ....................................................................................................................... 31
7
3.6 Movement of geminiviruses .......................................................................................................... 31
4. CONTROL OF GEMINIVIRUSES .............................................................................................................. 33
4.1 Management ................................................................................................................................ 33
4.2 Prospects for breeding for resistance ........................................................................................... 34
4.3 Prospects for genetic engineering for resistance .......................................................................... 35
REFERENCES ........................................................................................................................................... 37
CHAPTER TWO
MOLECULAR CHARACTERIZATION AND COMPARISON OF COTTON LEAF CRUMPLE
AND COTTON LEAF CURL GEMINIVIRUSES .............................................................................. 52
ABSTRACT ........................................................................................................................................... 52
INTRODUCTION ....................................................................................................................................... 53
MATERIALS AND MEllIODS ...................................................................................................................... 54
Virus isolates ..................................................................................................................................... 54
DNA extraction .............................................. .................................................................................... 55
PCR prinler design ............................................................................................................................ 55
PCR amplification of viral DNA ........................................................................................................ 56
Cloning of PCR fragments ................................................................................................................. 56
Southern hybridization .......................................... ............................................................................. 57
Nucleic Acid Sequencing ................................................................................................................... 58
Sequence analysis .............................................................................................................................. 58
REsULTS ................................................................................................................................................. 58
Total collon DNA extraction and PCR amplification of viral DNA ..................................................... 58
Size comparisoll of PCR amplified CLCrV alld CLCuV DNA ............................................................ 59
Differential hybridization ofCLCrV and CLCuV DNA A components ................................................ 59
Analysis of partial DNA A sequellces ofCLCrV and CLCuV .............................................................. 61
DISCUSSION ............................................................................................................................................ 62
REFERENCES ............. , ......................................................................................................................... 65
CHAPTER THREE
GENOME ORGANIZATION AND SEQUENCE ANALYSIS OF COTTON LEAF CRUMPLE
GEMINIVIRUS DNA A ....................................................................................................................... 77
8
ABSTRACT ........................................................................................................................................... 77
INTRODUCTION .................................................................................................................................. 77
MATERIALS AND METHODS •••••••...••••••.........•••••.•......•••••••••..••.••••••••••••.•.•......••..•••••.•.•••••....••••..........•....•••• 79
Virus isolate and cloned DNAs .......................................................................................................... 79
Nucleotide sequence determination ......................................... ........................................................... 79
Sequence analysis .............................................................................................................................. 80
REsULTS AND DISCUSSION ....................................................................................................................... 80
Nucleotide sequence and genome organi:ation ofCLCrV DNA A ...................................................... 80
Comparison of ell tire nucleotide sequellce ......................................................................................... 82
The intergenic region (IGR) ofCLCrV ............................................... ................................................ 82
Comparison of coat protein (CP) ORF ................................................... ............................................ 83
Comparison of replication associated protein (RAP. ACI) ORF ........................................................ 84
Comparison of AC2. AC3. AC4 ORFs ................................................................................................ 85
REFERENCES .••••••••.••••••••••...••••••••••••••.•.•..••••••••••..•.••.•••••••••••.•••••••••••••.•••••••••••••••••.••..••.•••••••.••.••.••.•.•....••.. 87
CHAPTER FOUR
GENOMIC SEQUENCE AND PHYLOGENETIC ANALYSIS OF COTTON LEAF CURL
GEMINIVIRUS DNA A ...................................................................................................................... 100
ABSTRACT .......................................................................................................................................... 100
INTRODUCTION ...................................................................................................................................... 100
MATERIALS AND METHODS ••••••••••••••.•.•......••••••••••.....•.•••••••••••••••••••••••••.•..••••••••••••...•.•••.•••••••••....•.......••.•• 102
Virus isolate and cloned DNAs .......................................................... ............................................... 102
Nucleotide sequence determinatioll ......................................... ...................................................... .... 102
Sequence alia lysis ............................................................................................................................. 103
Phylogelletic analysis ............................................................................................................ ........... 103
REsULTS A."ID DISCUSSION ...................................................................................................................... 1 03
Nucleotide sequellce alld ORFs ofCLCuV DNA A ............................................................................ 103
Conlparison of entire sequence ................................................................................................... ...... 105
The intergenic region (IGR) ofCLCuV ............................................................................................. 105
Comparison of AV I (precoal) sequences ........................................................................................... 106
Comparison of AV2 (capsid protein) sequences ................................................................................. 107
Comparisoll of ACI ORF .................................................................................................................. 107
9
AC2 ORF .......................................................................................................................................... 108
AC3 ORF .......................................................................................................................................... 108
AC4 ORF .......................................................................................................................................... 108
Phylogenetic analysis ............................................................................................................ ........... 109
REFERENCES .......................................................................................................................................... 11 0
10
List of Figures
CHAPTER 1
Fig. 1 Top Symptoms associated with cotton leaf crumple virus (CLCrV) Bottom Typical symptoms incited by cotton leaf curl virus (CLCuV) ..................................................................................................... 50
Fig. 2 Schematic of genome organization of type members of gemini virus groups ........................................................................................................... 5 I
CHAPTER 2
Fig. I
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
A generic genomic map of DNA A and locations ofPCR primers ......................................................................................................... 72
Alignment of the DNA sequences of the six bipartite whitefly transmitted gemini viruses .............................................................................. 73 Agarose gel electrophoresis ofPCR amplified DNA fragments from CLCrV ......................................................................................................... 74 Comparison ofPCR amplified DNA A fragments ofCLCrV and CLCuV ......................................................................................................... 74 Enhanced chemilluminscent Southern hybridization of CLCrV and CLCuV ......................................................................................................... 75 Alignment of the partial DNA sequences of the capsid protein gene region (A) and ALI gene region (B) ofCLCrV and
CLCuV ......................................................................................................... 76
CHAPTER 3
Fig. I Fig. 2 Fig.3A
Fig.3B
Fig.3C
Complete nucleotide sequence ofCLCrV DNA A. ........................................ 96 Proposed genomic organization for CLCrV DNA A. .................................... 97 Alignment of capsid protein partial sequences showing an imperfect zinc finger domain and an imperfect A TP-GTP binding motif ....................................... 98 Alignment of amino acids from 2 I 8 to 256, showing consensus of gemini viruses ................................................................................................. 98 Alignment of amino acids from 222 to 232 of ACI protein............ ......... ..... 99
CHAPTER 4
Fig. I Fig. 2 Fig.3A-G
Complete nucleotide sequence ofCLCuV DNA A. ..................................... I 17 Schematic ofCLCuV DNA A genome organization .................................... 118 Dendrograms showing the relationships of whitefly transmitted gemini viruses...................................................................................... I 19- I 20
11
List of Tables
CHAPTER 1
Table 1. Geminiviruses Transmitted by Bemisia tabaci ................................................ 47
Table 2 The functions of well characterized genes in subgroup III and their homologues in the other two subgroups ............................................................................ 48
CHAPTER 2
Table 1. Comparison of CLCu V and CLCrV partial sequences in AL 1 and CP regions with other geminiviruses ................................................................................ 68
CHAPTER 3
Table 1. Putative open reading frames (ORFs) in cotton leaf crumple (CLCry) DNA A .......................................................................................................... 92
Table 2. Amino acid sequence similarities and identities of CLCu V ORF with other geminiviruses ................................................................................................. 93
CHAPTER 4
Table 1. Putative open reading frames (ORFs) in CLCuV DNA A. ............................. 114
Table 2. Amino acid sequence similarities and identities ofCLCuV ORFs with other gemlnlvlruses............ .................................................................................. 115
12
ABSTRACT
Two whitefly transmitted cotton geminiviruses that differ in symptoms, but have
similar ecological properties, were characterized and compared at the molecular level. The
viruses, cotton leaf crumple (CLCrV) and cotton leaf curl (CLCuV) are found opposite
sides of the world, CLCrV in the western hemisphere, including southwestern US, Mexico
and Central America and CLCuV in Pakistan, other Asian countries and Africa During
recent years leaf curl has been a catastrophic disease being responsible for converting
Pakistan to net importer rather than exporter of cotton.
DNA A component of each virus was cloned with a polymerase chain reaction-based
technique. The complete sequence of CLCrV and CLCuV DNA A were determined to be
2630 and 2725 nucleotides, respectively. Sequence analysis show that CLCrV and
CLCuV CLCrV were most related to geminiviruses found in that part of the world from
which they originated. Thus CLCrV was most closely related to 'New World' viruses
such as belli1 dwarf mosaic and squash leaf curl viruses, while CLCuV was most closely
related to 'Old World' viruses such as Indian cassava mosaic and tomato yellow leaf curl.
CLCrV and CLCuV are least related to each other among the whitefly-transmitted
gemini viruses. Genome organizations predicted from the nucleic acid sequences and
phylogenetic analysis of whitefly-transmitted gemini viruses also reflect these relationship.
Chapter One
Introduction and Literature Review
1. Geminiviruses infecting cotton
13
Cotton (Gossypium hirsutum L.) is the most important fiber crop in the world. Grown
in countries on five continent, ~otton is a major crop in each of the five top producing
countries including China, USA, the countries of the former Soviet Union, Pakistan and
India (Hillocks, 1992). During 1992-93 global consumption of cotton roughly equaled
production of 86 million bales. Marked changes in production in major countries that
affect the import, export balance can have a negative impact on global trade. Such
changes in world production often occur because of plant disease epidemics.
Known diseases of cotton include 18 reported virus diseases (Watkins, 1981). Only
two, both whitefly-transmitted geminiviruses, have been reported as serious constraints on
cotton production in recent years in two of the major cotton producing countries, Pakistan
and the USA. When whitefly populations build up early in the season two separate
problems may result, sticky cotton from deposits of honeydew and viral infection. The
two viral diseases that threaten cotton production are cotton leaf crumple and cotton leaf
curl. Cotton leaf crumple virus (CLCrV) has been biologically characterized (Brown &
Nelson, 1984), and incites a disease of cotton in southwestern United States and northern
Mexico (Brown et al., 1987). Cotton leaf crumple is a disease characterized by floral
distortion, mosaic and general stunting, foliar malformation and yield losses in cotton (Fig.
1). It was first described in California in 1954 (Dickson et al., 1954) and in Arizona 1960
(Allen et al., 1960). The losses range from 21 to 76% (Brown et al., 1987). Geminate
particles, 17-20 nm in diameter, and dimers of 17-20 x 30-32 nm were visualized in viurs
preparations partially purified from CLCrV infected Red Kidney bean plants. CLCrV
14
infects numerous plant species in Malvaceae and Fabaceous families. Host range includes
both weeds and cultivated plants in Southwestern United States and Northern Mexico.
Year-round virus and/or vector reservoir exists in these cotton growing areas.
The other important viral disease of cotton is leaf curl. This disease has been reported
from Africa and Asia (Nour& Nour, 1963). The symptoms on cotton are different than
leaf crumple. Leaves of infected plants curl upward. There is a marked swelling of leaf
veins with enhanced dark green coloration. In addition, leaflike enations of various sizes,
which originated in the leaf nectary, can be found on lower side of the leaf. Infected plants
are stunted and distorted, bearing few flowers (Fig. 1). Yield may be reduced drastically
depending upon the time of infection. The disease is posing major production problems
for cotton in Pakistan (ICAC, 1993). In preliminary studies, geminivirus particles were
found associated with the diseased cotton plants (Hameed et al., 1994). While both types
of symptoms, leaf curl and leaf crumple, have been observed in cotton growing areas of
Pakistan, only CLCrV has been found in the USA. No useable cotton varieties are
resistant against these viruses although some breeding lines may show tolerance to
infection. The ultimate solution to the virus problem will be to genetically engineer cotton
with viral genes for resistance. This dissertation research was initiated to better
understand the molecular biology of the two viruses and to generate clones of viral genes
necessary for the genetic engineering.
2. Biology of gemini viruses
Both CLCrV and CLCu V belong to the geminivirus group. The group was
established in 1979 by the International Committee on the Taxonomy of Virus (ICTV)
based on a paired or geminate (twinned) particle morphology and a single stranded DNA
(ssDNA) genome (Matthews, 1979). The viruses in this group are transmitted by one or
several species of leafhoppers or whiteflies.
15
2.1 Symptomatology
Geminiviruses have many biological as well as physico-chemical properties in
common. One of the similarities in this group is the symptom expression. Goodman
(1981) grouped the geminiviruses into three broad categories on the basis of symptoms
they cause. The leafhopper transmitted geminiviruses of grasses, maize streak virus
(MSV) and chloris striate mosaic virus (CSMV), cause chlorotic streaks or striations on
the leaves of infected plants (Francki et al., 1979). The symptoms incited by these viruses
are different from those of other phloem-limited viruses of grasses, such as barely yellow
dwarf virus (BYDV), for which symptoms are most usually general yellowing (Goodman,
1981).
Geminiviruses that infect dicots are considered in two distinct groups. One group
causes leaf curl symptoms such as tobacco leaf curl virus ('foLCV) in tobacco, beet curly
top virus (Bcrv) in several of its many host species, and cassava latent virus in Nicotiana
clevelandii. The symptoms include curling and crinkling of leaf lamina, distortion of leaf
shape and reduction of leaf area, and in some cases formation of enations. Dwarfmg and
retardation of root growth are also commonly associated with the leaf curling symptom
complex (Goodman, 1981). The second type of symptom is the striking veinal chlorosis
or golden mosaic typical of dicots infected with viruses transmitted by whiteflies. Bean
golden mosaic in beans, the mosaic in Euphorbia pruni/olia, and tomato golden mosaic in
tomatoes are examples of this type. Plants infected by these viruses fIrst show vein
chlorosis that under some conditions lead to net like appearance, with bright yellow veins
and green interveinal areas. As the disease develops, the veinal chlorosis broadens into a
bright golden mosaic which under fIeld conditions is very obvious (Goodman, 1981). The
symptom expression may vary as influenced by speciflc plant host, conditions under which
plant is growing, stage of plant infected and the strain differences (Brown and Bird, 1992).
16
2.2 Morphology
Geminivirus virions are made of a pair of T=1 icosahedrals with a pentamer removed
from each icosahedron. The two icosahedrons are joined together where the pentamers
are removed. Each geminate particle consists of 110 capsid protein subunits of 29-30 kd
and one molecule of ss DNA (virion-sense) of 2.5 .... 3.0 kb (Goodman, 1977; Harrison et
al., 1977). The unique geminate particles are 20x30 nm in diameter. Thus the
morphology of the geminiviruses is unique among plant viruses described so far.
2.3 Transmission.
All the gemini viruses rely on Homopteran (Hemipteran) insect vectors for their
transmission. The insects feed by the way of stylet (Harrison, 1985; Stanley 1985).
Acquisition time, latent period, persistence in the vector, and inoculation time vary for
different virus-vector combinations. Generally, whitefly-transmitted geminiviruses are
acquired and transmitted less rapidly than the leafhopper transmitted viruses, which could
be a result of differences in insect feeding behavior (Harrison, 1985). Most viruses require
a latent period of four or more hours, and persist in their vectors for many days, possibly
even for life. There is no evidence for viral replication in their insect vectors (Cohen, et
al., 1989). The transmission is dependent only on acquisition period and viral
concentration in the plant sap (Boultan and Markham, 1986). Some viruses in this group
are sap-transmissible experimentally with ease (eg. bean golden mosaic virus (BGMV),
tomato golden mosaic virus (TGMV» (Brown and Bird, 1992). Markham et al. (1994)
compared 20 different colonies of B. tabaci including biotype 'B' and non -'B', from
different locations around the world for their ability to transmit more than 20
geminiviruses. All but two highly host specific colonies were capable of transmitting most
of the gemini viruses tested.
17
2.4 Geminivirus subgroups
Based on the number of genomic DNA, insect vectors, and host ranges, geminiviruses
are divided into three subgroups.
Subgroup I Subgroup II Subgroup III Number of ss DNA 1 1 2 Insect Vectors Leafhopper Leafhopper Whitefly Host Ranges Monocot Dicot Dicot Type Member Maize streak Beet curly top Bean golden
virus virus mosaic virus
2.5 Evolutionary relationship.
The relationship between the geminiviruses has been studied at the level of DNA
sequence, protein sequence, serological cross reactivity and restriction site polymorphism
(Howarth & VandeMark 1989; Pinner & Markham, 1990; Pinner et al., 1992; Rybicki,
1991, Rybicki & Hughes 1990; Hughes et al., 1992; Timmermans et al., 1994). A
phylogenetic tree based on coat protein and replication associated protein sequences,
shows that there is a greater sequence divergence of monopartite geminiviruses compared
to bipartite geminiviruses (fimmermans et al., 1994). This observation is consistent with
the fact that bipartite gemini viruses are serologically more closely related than
monopartites, which show limited cross reactivity (Harrison, 1985; Stanley, 1985).
Therefore it could be implied that monopartite monocot-infecting viruses may be ancestral
to the bipartite viruses. Timmermans et al. (1994) proposed three steps for this
transition: infection of dicots, acquisition and whitefly transmission, and acquisition of a
bipartite genome. A tentative order of these events was observed by Timmermans and
Messing (unpublished) in their phylogenetic analysis of open reading frame (ORF)
sequences. Viruses like tobacco yellow dwarf virus (Tob YDV), BCTV, tomato leaf curl
18
(TI..-CV) and tomato yellow leaf curl (TYLCV) are possible intennediates in the transition
between monopartite to bipartite. This transition might have occurred because of
duplication of DNA A, since there is a considerable homology between A VI and BVI
(Kikuno et al., 1984). Mutational analysis of the CP region of different geminiviruses
further supports this DNA duplication hypothesis. Geminiviruses with a single genomic
DNA have no capacity to cause disease in the absence of CP (Lazarowitz et al., 1989;
Boulton et al., 1989; Briddon et al., 1989). Systemic infection by geminiviruses with
bipartite genomes is not severely affected by disruption of CP (Brough et al., 1988;
Etessami et al., 1989) whereas mutation in the CP cistron resulted in delayed and
decreased symptoms in TLCV and TYLCV-Th (Rigden et al., 1993; Rochester et al.,
1994). These viruses are placed among the intermediate geminiviruses.
Geographical isolation may have a role in the evolution of geminiviruses (Howarth &
VandeMark, 1989). Thus African cassava mosaic (ACMV), TLCV and TYLCV from the
Old World and form their own branch of the phylogenetic tree separated from the New
World geminiviruses (fimmermans et al., 1994, Chapter 4). Frischmuth et al. (1993)
demonstrated the evolutionary divergence of Old World and New World geminiviruses by
constructing pseudorecombinants among the genomic DNA A and DNA B of ACMV,
Indian cassava mosaic (lCMV), Abutilon mosaic virus (AbMV), and TGMV. ACMV
DNA B was able to mediate the systemic movement of ICMV, TGMV and AbMV DNA
A components. In reciprocal experiments DNA B of neither TGMV nor AbMV could
mediate the systemic movement of ACMV DNA A although DNA B of TGMV and
AbMV can support the movement of each other's DNA A. This variation in movement
protein (MP) specificity suggests evolutionary divergence of Old and New world
gemini viruses.
3. Molecular biology of gemini viruses
3.1 Genome organization
The three subgroups of the gemini viruses have different genome organizations,
reflecting the biological diversity of the geminiviruses. However, several features are
conserved across the diverse geminiviruses. These conserved features are listed below,
followed by the differences among different subgroups of geminiviruses.
• Genes are encoded in both the virion-sense and the complementary sense DNA.
19
• Existence of an intergenic region (IGR) between the 1st ORFs of the virion-sense
DNA and the complementary DNA. IGR is identical in the DNA A and DNA B of
a geminivirus, but is different among different geminiviruses. A nonanuc1eotide
(T AA T A IT AC) within IGR is conserved in all geminiviruses and is positioned at
the loop region of a stable stem-loop predicted in all geminiviruses.
• A capsid protein gene is encoded on the virion sense DNA and replication related
proteins are encoded on the complementary sense DNA.
3.1.1 Subgroup 1
Members of this subgroup are transmitted by leafhoppers, have a monopartite genome
and infect monocotyledonous plants. Among its members are maize streak virus (MSV)
(Howell, 1984; Lazarowitz, 1988; Mullineaux et 01., 1990), WDV (MacDowell et 01.,
1985; Woolston et 01., 1988), and panicum streak virus (PSV) (Briddon et 01., 1990).
The genomes of these viruses contains four ORFs and two non-coding regions (Fig. 2).
These ORFs diverge from the larger intergenic region and converge at the smaller
intergenic region. The ORFs VI and V2 are on the viral strand. The ORF VI has clearly
been shown to function in the virus movement (Boulton et at., 1993), while V2 encodes
20
for coat protein and is required for systemic movement, vector transmission and disease
development of the virus (Lazarowitz et al., 1989). The genome organization on the
complementary-sense DNA is quite different from other geminiviruses. There are two
large ORFs (Cl and C2). Amino acid sequences predicted from these two ORFs show
considerable degree of homology with that of ACI gene in subgroup ill geminiviruses.
Cl is homologous to the N-terminal portion of the ACI protein and C2 is homologous to
the C-terminal portion of the ACI protein. ORF C2 in most of the monopartite
gemini viruses does not contain A TG initiation codon (Andersen et al., 1988; Briddon et
al., 1992, Donson et al., 1988; Hughes et al., 1993; MacDowell et al., 1985). The Cl
and C2 are expressed as a single ORF via a splicing event Such splicing has been
confmned for WDV (Dekker et al., 1991; Schalk et al., 1989) and Digitaria streak virus
(DSV) (Accotto et al., 1989; Mullineaux et al., 1990).
3.1.2 Subgroup 11:
This group of leafhopper transmitted, dicot-infecting viruses has only one genomic
DNA. Beet curly top virus (BCfV) is the only member of this group (Stanley et al.,
1986). BCfV is differentiated from subgroup I on the basis of unique genome
organization (Fig. 2) (Stanley et al., 1986) and ability to infect a wide variety of dicot
plant hosts (Bennett, 1971). Stanley et al. (1986) demonstrated that the single DNA
component of California strain of BCTV contained all viral information necessary for
replication, systemic movement, encapsidation, symptom production, and transmission by
leafhoppers. Of the seven ORFs conserved in three sequenced strains ofBCTV, (Stanley
et al., 1986; Stenger et al., 1990; Stenger, 1994), three are encoded by virion strand
(ORFs VI, V2 and V3) while four are encoded by complimentary strand (ORFs Cl, C2,
C3 and C4). Mutational analysis suggested that each of the seven ORFs are functional.
21
Briddon et al. (1989) detennined that ORF Cl was essential for replication and
infectivity. BCfV capsid protein (ORF VI) is required for systemic movement (Briddon
et al., 1989) and also mediates transmission of the virus by leafhoppers (Briddon et al.,
1990). BCfV ORF V3 has been identified as a novel geminivirus gene required for
efficient movement (Hormuzdi & Bisaro, 1993; Frischmuth et al., 1993), while another
virion sense ORF (V2) has been associated with the accumulation of ssDNA (Stanley et
al., 1992; Hormuzdi & Bisaro, 1993). Altered symptom expression associated with
mutations in BCfV ORFs C2, C3, and C4 (Stanley & Latham, 1992; Stanley et al., 1992)
provide evidence that these ORFs are functional, but the primary functions are not yet
known.
3.1.3 Subgroup 111
Viral genomes in this subgroup consist of two ssDNA components, designated as A
and B. Each of these DNA molecules is encapsidated in a separate geminate particle,
requiring double inoculation of both DNA components for successful infection (Goodman
et al., 1980). Bipartite viruses are whitefly-transmitted and infect dicots. This is the
largest subgroup with more than 50 viruses described so far (Markham et al., 1994). List
of these viruses and accepted code by ICfV is given in Table 1. Viruses such as TGMV,
BGMV and Abutilon mosaic virus (AbMV) are among the members of this subgroup.
Genes are arranged bidirectionally on each DNA component (Lazarowitz, 1992). DNA A
encodes in the virion strand a single, large ORF for the capsid protein (AVl) (Lazarowitz,
1992). In the complementary strand, DNA A encodes four ORFs (ACl, AC2, AC3, AC4)
(Lazarowitz, 1992). All the proteins essential for the replication of this gemini virus
subgroup are encoded on the DNA A. DNA B contains two large ORFs, one each on the
virion-sense DNA and on the complementary sense DNA. These two ORFs encode
22
proteins necessary for the movement of virus in infected plants. Recent analysis of
nucleotide sequences of dicot geminiviruses help identify iterative sequence motifs of 8-12
nucleotide in the lOR. Organization of these sequences, number, situation and spacing is
highly conserved within three major lineages of dicot geminiviruses (Arguello et al., 1994).
The functions of well characterized genes in subgroup III and their homologues in the
other two subgroups are summarized in Table 2.
Several geminiviruses have properties intermediate to the subgroups described above.
Some isolates of two recently reported viruses, TYLCV and TLCV, appear to fall
between subgroup II and subgroup Ill. Isolates from TYLCY for Israel (Navot et al.,
1991) and Sardinia (Kheyr-pour, 1992) appear to have single DNA components, while an
isolate from Thailand (Rochester et al., 1994) and some other places contains two DNA
components. DNA B in some isolates of TYLCY is not required for the virus movement
and symptom expression (Rochester et al., 1994). Another virus TobYDY transmitted by
leafhopper, features the genome organization similar to monocot geminiviruses in
subgroup I, such as two intergenic regions, four ORFs and an intron (Morris et al.,1992),
but have adapted to dicot hosts.
3.2 Subgenomic DNA
Several of the geminiviruses are associated with small defective genomic components
(Stanley & Townsend, 1985; Frischmuth & Stanley, 1992; MacDonalds et al., 1988;
Saunders et al., 1991). These genomic components depend on the parent virus for their
proliferation. Both ssDNA and double stranded DNA (dsDNA) forms of subgenomic
DNA exist at a low concentration in infected tissue and the ssDNA form is encapsidated
(Stanley, 1983). The subgenomic DNA has been characterized and shown to comprise a
family of closely related molecules that derive solely from a specific region of DNA B
(Frischmuth & Stanley, 1993). The deletions within this genomic component serve to
remove gene BVI entirely and disrupt the carboxy-tenninus of gene BCI. Preliminary
experiments in which cloned copies of genomic and subgenomic components of ACMV
were co-inoculated onto N. benthamiana suggested that subgenomic DNA disrupted
virus proliferation and behaved as defective interfering DI DNA (Stanley & Townsend,
1985).
3.3 Replication
3.3.1 Site of replication
23
Geminivirus replication is confined to the cell nucleus. Viral like particles accumulate
in the nuclei of infected plants. Cytopathological changes that have been associated with
geminivirus infection have been primarily in nuclear or nucleolar structures (Goodman,
1981). Davies and Stanley (1989) observed irregular aggregates or hexagonal crystalline
arrays of virus particles in the nuclei of infected plants. Major cytopathological changes in
dicot specific geminiviruses have been observed (Adejare & Coutts, 1982; Kim et a/.,
1978), which include repositioning of chromatin to the vicinity of nuclear membrane,
hypertrophy of the nucleoli, and segregation of nucleolar components into separate
granular and fibrillar regions. These changes may reflect the shift from host to viral
transcript synthesis (Kim et a/., 1978). Infection is also accompanied by the appearance of
fibrillar rings in the nucleus (Esau, 1977; Kim et a/., 1978). These rings consist primarily
of DNA and protein, and their appearance seem to coincide with the appearance of viral
particles, they may be the site of viral DNA synthesis and viral assembly (Kim et a/.,
1978).
3.3.2 Replication mechanism
The mechanism of viral DNA replication also distinguishes geminiviruses from all
other plant viruses. Geminiviruses replicate through a dsDNA intermediate in the nucleus
24
of the infected cells. A rolling-circle model for geminivirus replication analogous to the
replication of the bacterial coliphage cpX174 and a Staphylococcus aureus (M 13) ssDNA
plasmid has been proposed Evidence consistent with the rolling-circle replication
mechanism has been reported for Bcrv (Stenger et al., 1991), ACMV (Saunders et al.,
1991), and WDV (Heyraud et al., 1993). ACI is the only viral protein absolutely required
for viral DNA synthesis (Elmer et al., 1988; Etessami et al., 1988), and may initiate this
process by introducing a nick at the origin of replication. It may also facilitate the binding
of host proteins to the replication origin, a function provided by the large T antigen of the
SV40 during its replication (Fontes et al., 1994). ACI protein interacts specifically with
sequences in the IGR (Fontes et al., 1994) near the transcription initiation sites ofBCl
gene and ACI gene.
3.3.3 Complementary strand Synthesis
Following the uncoating of the virion ssDNA, the first step is the synthesis of the
complementary strand from ssDNA template leading to the production of transcriptionally
active ds DNA intermediates. This complementary DNA synthesis is accomplished
entirely by host proteins since naked ssDNA is infectious when introduced into plants.
This synthesis is primed by a short stretch of nucleotides complementary to nucleotides in
the IGR.
In the monocot-infecting geminiviruses (subgroup I), a short complementary strand
DNA fragment is encapsidated in the virions. The DNA fragments of MSV (Dons on et
al., 1984) and WDV (Hayes et al., 1988) are about 80 nucleotides containing defined 5'
terminal end and heterogeneous 3' end. Such primers within subgroup TIl and TI have not
been found in virions and they may be synthesized de novo immediately after the uncoating
of the viral ssDNA. An RNA primer preceeding the synthesis of the complementary DNA
25
of ACMV has been identified in the DNA replication intennediates from ACMV infected
tobacco plants (Saunders et al., 1992).
3.3.4 Synthesis of virion strand
The following synopsis of the virion strand DNA synthesis is largely derived from
excellent studies on African cassava mosaic virus (ACMV) (Saunders et al., 1992) and
wheat dwarf virus (Heyraud et al., 1993b).
The dsDNA serves both as a template for transcription and as a template for rolling
circle replication. Transcription from the dsDNA and subsequent translation produce
virus encoded proteins (ACl/Cl) which are required to initiate replication. The intergenic
region of the circular single-stranded DNA genome of geminiviruses contains a sequence
which can potentially be able to fold into a stem-loop structure. This sequence may serve
as the origin for rolling-circle replication. At the onset of virion-sense DNA synthesis, a
nick is introduced in the closed circular dsDNA at the origin of replication. ACl/CI
proteins have been proposed as the sequence-specific nuclease; however, this function of
the ACl/CI proteins have yet to be demonstrated experimentally. On the basis of
homology with the gene A cleavage site of ~X174 phage, the nicking site has been
suggested within the nonanucleotide conserved in all geminiviruses. Consistent with this
hypothesis, Heyraud et al. (1993 a,b) reported the nicking within the TACCC sequence of
the conserved nonanucleotide of WDV. The 3' terminus of the nicked DNA serves as a
primer for DNA synthesis, displacing the original virion-sense DNA as the template
(complementary) strand is copied. The enzyme(s) that synthesize the virion strand
continuously circle around the complementary DNA template, hence, the rolling-circle
replication model. As a unit-length virion strand is synthesized, it is cut and ligated to
fonn a close circular ss virion DNA. This close circle ssDNA can either serve as template
for another round of replication or can be encapsidated into virions. If the unit-length
virion DNA is not released immediately, concatmers of the viral DNA is fonned.
26
In addition to an endonuclease activity, viral DNA synthesis via rolling circle require
helicase and ligase activities. Presence of ligase activity has not been addressed in
geminiviruses, but a helicase activity has been ascribed to ACI protein. ACI protein binds
to elements in the IGR 4 folds stronger with ssDNA than dsDNA, a property common to
many DNA helicases (fhommes et al., 1993). Protein sequence comparison has also
identified an NTP binding site as part of a putative helicase domain at the C terminus of
ACI and in ORF C2 of the CI-C2 protein (Gorbalenya et al., 1990).
3.4 Gene expression
3.4.1 Transcription
Bidirectional transcription from the common region or large IGR has been shown for
bipartite (Frischmuth et al., 1991; Petty et al., 1988; Sunter et al., 1989) and monopartite
geminiviruses (Accotto et al., 1989; Dekker et al., 1991) respectively. Transcripts from
each strand are 3' co-tenninal, but heterogenous at the 5' end. A variety of mapping
techniques have identified several types of transcription units. One type, resembling a
typical eukaryotic mRNA, is flanked by a conventional TAT A box and polyadenylation
signal and encodes a single protein (eg. viral strand transcript of bipartite viruses). A
second type is also flanked by the conventional expression signal, but is polycistronic (eg.
complementary strand transcript of DNA A of bipartite genomes). Some transcripts are
not flanked by appropriate 5' transcriptional signals and may arise from cleavage of a
longer precursor (eg. transcript 2 of DNA B of bipartite geminiviruses). Transcription
maps of different viruses within a subgroup resemble each other, but some differences
exist (Timmermans et al., 1994). Unlike ACMV, in which two overlapping transcripts of
27
1.6 and 0.7 kb were mapped (Frischmuth et al. 1991), both TGMV (Sunter & Bisaro,
1989; Sunter et al., 1989) and AbMV (Frischmuth et al., 1991) produce a 1 kb transcript
from the complementary strand of DNA A. In vitro translation products of RNA
transcripts of TGMV were analyzed by Thommes & Buck (1994). The results of the
experiments suggested that the DNA A of whitefly transmitted (WFI) geminiviruses may
be expressed by two principal mRNAs, one encoding the ACI and AC4 proteins and et
al., 1985) and DSV (other encoding the AC2 and AC3 proteins.
Only a single dicistronic transcript is produced from the viral strand of WDV (Dekker
et al., 1991), but MSV (Morris Accotto et al., 1989) produce a second shorter transcript
encoding the coat protein, probably from cleavage of the larger one.
3.4.2 Regulations of gene expression
Temporal regulation of gene expression is a characteristic feature of many viruses.
Evidence for such regulation in gemini viruses exits as well. The promoters of bipartite
virus genes that are required late in infection are weak in transient assays (Zhan et al.,
1991), but are activated late in the infection cycle by an early gene product encoded by
AC2 (Haley et al., 1992; Sunter and Bisaro, 1991, 1992). In addition, the replication
associated protein (RAP, AC1) of ACMV and TGMV may repress its own expression
later in the infection (Haley et at., 1992; Sunter et al., 1993). Transcription of B genome
should be affected because it shares common region with the A genome,that contains the
transcriptional promoter. Such reduction was shown for the BCI gene of ACMV (Haley
et al., 1992). This was not the case for TGMV, possibly because another downstream
transcription start site is used (Sunter et al., 1993).
Temporal control of gene expression in monopartite gemini viruses has also been
reported. Expression of the viral strand genes is controlled partly by an "early" gene
28
product, the CI-C2 protein (Hofer et al., 1992). Differential splicing may also regulate
viral gene expression (Accotto et al., 1989; Dekker et al., 1991; Mullineaux et al., 1990;
Schalk et al., 1989). Both splicing of the viral transcripts and translational ribosomal
frameshifting have been suggested in the gene expression of this subgroup. A single
mRNA containing the two ORFs predicted on the complementary strand of wheat dwarf
virus is posttranscriptionally processed by specific splicing (Schalk et aI., 1989). An 86 bp
intron located in the overlapping region of the two ORFs is spliced to fuse the two ORFs
into a single ORF, thus expressing a protein of 41 kd, homologous to the ACI protein of
the subgroup ill geminiviruses. This posttranscriptional splicing may be differentially
regulated such that two proteins, one from un spliced RNA and one from the spliced RNA
can be expressed from a single transcriptional event
Dekker et al. (1991) showed that WDV-CJI uses two different mechanisms for
expressing overlapping open reading frames (ORFs). Mapping of the virion sense RNAs
identified a single polyadenylated transcript of 1.1 kb spanning the overlapping ORFs VI
and V2 that encode cell-cell spread functions and the coat protein respectively. This
finding distinguishes WDV from other monocot-infecting geminiviruses studied so far
which were shown to encode two 3' co-terminal transcripts capable of expressing either
the VI or V2 ORF. A survey of codon usage at the junction between the VI and V2 ORF
has led them to propose that translational frame shifting analogous to that in the yeast Ty
element may occur. Analysis of polymerase chain reaction (PCR) amplified
complementary sense eDNA clones, however has revealed the presence of mature spliced
and un spliced RNAs which could encode products of an intron mediated Cl:C2 ORF
fusion or the Cl ORF product alone.
In the only carefully studied virus, MSV, the promoter element for the virion-sense
gene transcription is identified as two GC-rich repeats resembling the Spl binding sites
found in the promoters of genes in animal cells and viruses (Fenoll et al., 1990). This
element appears also to bind a nuclear factor.
3.5. Function of the viral genes
3.5.1 Function of the AC11C1 protein
29
AC1 appears to have a dual function. This protein can bind to a specific sequence in
the IGR to initiate synthesis of virion strand ssDNA and to repress its own transcription.
Sunter et al. (1993) replaced the AC1 and BC1 ORF ofTGMV with the ~ glucuronidase
(GUS) reporter gene and used the gene replacement constructs to examine AC1 and BC1
gene expression in tobacco protoplasts. They found that expression of the GUS reporter
in the AC1 replacement construct was reduced to background levels when transfections
included a plasmid expressing ACl protein from the cauliflower mosaic virus 35S
promoter, indicating that AC1 gene expression is autoregulated. Surprisingly, a similar
repression of BC1 gene expression by AC1 protein was not observed.
Two recent studies showed that AC1 protein is a sequence-specific protein that binds
to the sequence within the IGR. A 13 bp element (GGTAGTAAGGTAG) in the
intergenic region constitutes a high affinity binding site. DNAs containing mutations in
these sequence motifs are not able to replicate (Fontes et al., 1994). The AC1 binding site
also overlaps with the transcription initiation site of AC1 gene (Fontes et al., 1994; Eagle
et al., 1994). These studies together provide a model for the autoregulation of AC1
gene. Upon the infection and the subsequent dsDNA formation, AC1 gene is expressed to
facilitate the replication of the viral DNA. AC1 protein then binds to the IGR to initiate
the replication of viral RNA. This binding also occupies the transcription initiation site
and thus shutdown its own expression.
30
Fontes et al. (1992) showed that ACI ofTGMV interacts specifically with A and B
DNA by using an immunoprecipitation assay for AC1:DNA complex formation. In this
assay, a monoclonal antibody against ACI precipitated AC1:TGMV DNA complexes,
whereas an unrelated antibody failed to precipitate the complexes. Competition assays
with homologous and heterologous DNAs established the specificity of AC1:DNA
binding. ACI produced by transgenic tobacco plants and by baculovirus-infected insect
cells exhibited similar DNA binding activity. The ACI binding site was maped to 52 bp on
the left side of the common region, a 235-bp region that is highly conserved between the
two TGMV genome components. The ACI :DNA binding site does not include the
putative hairpin structure that is conserved in the common regions or the equivalent 5'
intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus
replication protein is a sequence-specific DNA binding protein.
Lazarowitz et al. (1992) investigated interactions between ACI and the IGR of
TGMV and squash leaf curl (SLCV) demonstrating selectivity and sequence specificity in
this protein-DNA interaction. Simple component switching between the DNAs of TGMV
and SLCV and analysis of replication in leaf discs showed that whereas the A components
of both TGMV and SLCV promote their own replication and that of their cognate B
component, neither replicates the noncognate B component. Furthermore, using an in
vivo functional replication assay, Lazarowitz et al. (1992) found that cloned viral IGR
sequences function as a replication origin and direct the replication of non viral sequences
in the presence of AC1, with both circular single-stranded and double-stranded DNA
being synthesized. Finally, by the creation of chimeric viral common regions and specific
subfragments of the viral CR, they demonstrated sequence-specific recognition of the
replication origin by the ACI protein, thereby localizing the origin to an approximately 90
nucleotide segment in the ACI proximal side of the CR that includes the conserved
31
gemini viral stem-loop structure and approximately 60 nucleotides of 5' upstream
sequence. These studies provide another evidence that ACI is a sequence specific binding
protein that is required for replication.
3.5.2 Function of AC2 protein
The function of AC2 is to activate the expression of late genes including AVI (capsid
protein gene) and BVI (movement protein gene) ..
Sunter et al. (1992) have investigated the role of the AC2 protein in the regulation of
A and B component gene expression in TGMV and examined the transcriptional and post
transcriptional components of this regulation. They found that AC2 protein is required for
efficient expression of both the A VI and BVI genes, but not the BCl gene. A
comparison of steady-state transcript levels and transcript levels determined by nuclear
run-on analysis showed that activation of AVI and BVI gene expression by the AC2
protein occurs primarily at the level of transcription. These results provide an explanation
for the lack of infectivity demonstrated by AC2 mutants, and suggest that the AC2 protein
interacts with the cellular transcription machinery to activate the expression of rightward
viral genes.
3.6 Movement of gemini viruses
In general, subgroup ill geminivirus DNA A is capable of replicating in protoplasts or
in leaf disks but unable to infect whole plants, thus the DNA B of this group of viruses
may encode functions necessary for cell-to-cell movement and long distance transport
Ingham & Lazarowitz (1993) identified a mutation within the BVI gene responsible for
inability of SLCV to infect Nicotiana benthamiana, a host for the wild type virus. The
mutation was identified as a substitution of cysteine for arginine at position 98 of the BVI
protein. The mutant replicates to the wild type level in the infected protoplasts and retains
32
infectivity for pumpkin, squash, and bean plants. Using a baculovirus expression system
and immunogold cytochemistry, Pascal et af. (1994) established the functions of BCl and
BVI proteins of SLCV. BVllocalize in the nucleus and BCl in plasma membrane which
suggests that BVI is involved in the shuttling of the genome in and lor out of the nucleus,
whereas BCl acts at the plasma membrane cell wall to facilitate viral cell-to-cell
movement.
In a related study, Pascal et al. (1993) expressed both BCl and BVI of SLCV in
transgenic plants. Their results showed that the expression of BCl alone is sufficient to
cause mosaic and leaf curl symptoms, typical of SLCV infection. The expression of BVI
had no effect on plant growth and development Kim and Lee (1992) found tubular
structures associated with Euphorbia mosaic virus infection and suggested that they are
involved in the cell-to-cell movement of this virus in the early stage of the infection. Cells
of the earliest chlorotic lesions in mechanically inoculated leaves revealed cytopathic
effects that were not observed in systemically infected leaves or in advanced lesions of
inoculated leaves. These were the occurrence of macrotubules containing geminate virus
like particles in the cytoplasm and continuation of the macrotubules with the
plasmodesmata. No virus like particles occurred in the plasmodesmata of cells that were
apparently without macrotubules, suggesting that the presence of the macrotubules is
required for the occurrence of virus like particles in the plasmodesmata. The nuclei of
cells that had macrotubules contained characteristic nucleopathic effects of geminivirus
infection, indicating that the macrotubules were virally-induced.
A molecular genetic analysis of the DNA B of genes of BDMV was carried out using
full-length infectious DNA A and DNA B clones of BDMV. Frameshift mutations or
single amino acid substitutions in either of the BCl or BVI ORFs abolished the systemic
movement of the virus, but did not affect DNA B replication in protoplasts. The specific
role of these proteins was established as BCI protein was shown to move from cell-to
cell, increase mesophyll plasmodial size exclusion limit, and potentiate the movement of
double-stranded DNA from cell-to-cell. Movement of single and double-stranded DNA
out of the nucleus is mediated by BVI protein. This provides the direct experimental
evidence for intercellular macromolecular transport in plants, and suggests that the BVI
and BCI proteins coordinate the movement of viral DNA across both nuclear and
plasmodesmal boundries (Noueiry et al., 1994). This apparently is conflicting with the
data reported by Kim and Lee who suggested that geminiviruses move as virions from
cell-to-cell.
33
Even more surprising are findings that Abutilon mosaic virus (AbMV) and ACMV
DNA A can systemically infect N. benthamiana plants, although at a low level. DNA B,
which contains all the movement protein genes increase the rate of the virus spread, but is
not essential for systemic infection. These results suggests that the DNA A by itself is
capable of cell-to-cell movement and long distance movement and the movement proteins
only improve the efficiency of the virus movement The necessity of the movement
proteins for systemic infection and the role they play in the viral infection are still
unanswered.
4. Control of gemini viruses
4.1 Management
Management is a broad term referring to the process of taking advantage of cultural
practices with the crops that reduce the potential of damaging epidemics from viral
diseases. One of the classic programs in this category aimed at a gemini virus is the beet
curly top program in place in coastal California valleys for the past 50 years (Duffus,
1983). This program uses a variety of strategies that include mature plant tolerance,
manipUlation of planting dates to avoid vector populations, reduction of vector
populations in natural habitats during the off season and avoidance of areas for tht;
planting of sugar beets with frequent recurring epidemics.
34
Another recent management plan was developed where advanced spatial analysis
information processing technology (geographic information systems and geostatistsics)
was used to identify areas of recurring epidemics in a WFf gemini virus complex of
tomatoes in the The Del Fuerte Valley of Northern Sinaloa, Mexico. This information was
used to avoid known infection hazards such as overlapping crops, urban landscaping
plants and weeds that played a role either as a vector host or a source of virus (Nelson et
al., 1994). One approach that clearly does not work in a management plan is the use of
insecticides to control the vector after virus symptoms appear in the crop (Bolkan and
Reinert, 1994). Year-round management of vector and virus sources is the proper cultural
management approach to impact jointly on the vector populations and the virus sources.
4.2 Prospects for breeding for resistance
Plant genes involved in virus resistance are poorly understood, therefore conventional
breeding programs are often difficult, time consuming and frequently ineffective. For
example, resistance to cassava mosaic disease is polygenic and recessive. The cultivars
considered to be resistant are ii1 fact susceptible to ACMV infection (Hahn et al., 1980).
Cotton breeding lines are available that show a lack of severe symptoms when infected by
CLCrV. These lines, however, do not have productive commercial potential in the present
form (Brown & Wilson, 1991). Tomato varieties resistant to TYLeV have been
developed in Israel. Because the varieties lack some cultural characteristics desired by
growers, a crop free period is still used to control the virus disease (personal
communication, Shlomo Cohen).
Some sugar beet varieties have long had mature plant resistance to BCTV that in
conjunction with cultural management strategies protects the very young plants and makes
a useful component of the current management plan. Tomato breeding lines resistant to
curly top have been available for 30 years but cultural management strategies are still
preferred.
4.3 Prospects for genetic engineering for resistance
35
The alternative approach to make plants resistant to virus infection is using pathogen
derived resistance. Most of the effort has been focused on RNA viruses (for reviews see
Hemenway et al., 1990; Wilson, 1993). Basically, three approaches have been used to
engineer plants for viral resistance. One is to explo~t the ability of satellite RNA to
interfere with the viral replication. The second strategy is based expression of viral
proteins in transgenic plants that confer resistance to related and more virulent viruses.
The third method uses the antisense RNA expression.
Similar approaches have been used for geminiviruses. Transformation of N.
benthamiana with sub genomic DNA B of ACMV and subsequent challenge with wild
type virus showed ameliorated symptoms and reduced viral DNA level for isolates of
ACMV and 60% reduction in full length DNA B (Stanley et al., 1990). Subgenomic
DNAs are associated with many geminiviruses (Frischmuth & Stanley, 1992; Kammann et
al., 1991; MacDowell et aI., 1986; MacDonald et al., 1988; Stenger et al., 1992). This
method, however, would have very limited use because of the specificity of the RAP
(Stanley et al., 1990). Natural cross protection has not been observed between
geminiviral isolates for unknown reasons (Stanley et aI., 1990).
The antisense approach has been successful for engineering protection against TGMV
(Day et al., 1991). Transgenic tobacco plants expressing antisense RNA ofTGMV ACI
ORF showed a drastic reduction in symptom development upon agroinfection with
TGMV. Viral replication was inhibited in these plants, and antisense abundance was
correlated with viral DNA levels and resistance. These plants were also tested for
suppression of replication of ACMV and BCIV, which bear 64% and 63% overall
nucleotide sequence homology to TGMV, respectively (Bejarano & Lichtenstein, 1994).
Using agroinfection of leaf disks, under the conditions in which TGMV replication was
reduced lO-fold, BCIV replication was reduced 4-fold, and ACMV replication
unaffected. The fact that TGMV is homologus to BCTV in stretches of AC1 and
homology with ACMV is more evenly distributed may explain their observations. The
antisense approach may be more successful for geminiviruses that have ssDNA genomes
located in the nucleus than for RNA viruses with life cycle restricted to cytoplasm.
36
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47
Table 1 Geminiviruses Transmitted by Bemisia tabaci
Virus *Code Country of origin _EUPhO(bla mosruc EuMV N. menca Texas nenner lPGV N. menca Sauash leaf curl SL :V menca
otton leaf crumole L rV menca omato mottle TMoV menca elon leaf curl MU V menca atermelon curly mottle WCMV menca Issadula mosruc WMV . A.menca
hynchoSla mosruc KhMV PuertoRlco Passltlora leaf mottle PLM Puerto Rico Malvaceous chloroSIS MeV Brazil. Puerto Rico Peoner mIld til!re PeMTV MeXICO Chino del tomate CdTV MeXICO Til!re disease TDV MeXICO
maloa tomato leaf curl STL( V MeXICO ean Calico mosruc BCaMV menca ida I!olden mosruc SiGMV menca
Jatrooha mosruc JMV menca Tomato yellow. mosruc ToYMV menca Tomato I!olden mosruc TGMV menca Hean I!olden mosruc BGMV menca Bean dwarf mosruc BDMV · Amenca
~E~uo~hlo~r~bim~m~o~s~ru~c~ __________ ~EuMV Potato yellow mosatc PYMV
· Amenca · Amenca
Abutllon moSatc AbMV · Amenca Solanum aOlcalleat curl SAL( V · Amenca El!l!olant yellow mosaic EYMV · Amenca Tomato yellow leat curl TYLeV · Amenca. Afnca ASia Australia "tomato leat curl TLCV MeddJteraman Jaoan Sudan. India Tobacco leal curl 10LCV Australia Watermelon chlorotiC stunt WcsV ASia. Yemen Kenya Pseuderantheum yellow vem PYVV Yemen Tomato yellow dwarf ToYDV Yemen Ghana Sml!aoore. Honeysuckle yellow vem mosatc HYVMV l eylon Japan Eupatonum yellow vem I:!pYVY Japan Munl!bean yellow mosaic MYMV Japan Soybean cnnlde leat SC :1 ASia Horsel!ram yellow mosaIc lil!Y MY ASJa Lupm leat curl L Jl V India Indian cassava mosruc I M V India Atncan cassava mosruc Al MY Atnca Basil I!olden moSatc BaGMY Kenya
ottonIeat curl CLl uV Sudan. Pakistan .... owpea I!olden mosatc CGMY r:. Alnca W. Athca Ida vellow vem SYW
Macrotvloma mosaIc MMV Benm Llmabean I!olden mosruc LGIVl Asvstasla I!O den mosatc AGM V Benm Nil!ena Doltchos vellow mosaIc Do Y IV India Okra leaf curl 01 Bemn Al!eratum ve low vemAYY II ASia, Sinl!aoore
* Accepted code specified by the internaJiofUll Committee on Taxonomy of IIi ruses
48
Table 2
The functions of well characterized genes in subgroup m and their homologues in
the other two subgroups
Genes Aprox. Functions (possible)
Sizes
AC1 40kd Replication essential, autosuppressor, helicase, site-specific
nuclease.
AC2 15 .... 20 kd Transactivator for AV1 and BV1 gene expression
AC3 14 .... 16 kd DNA accumulation, replication efficiency
AC4 15 kd Disease symptom, AC1 gene regulation, movement
AV1 27 .... 30 kd Capsid protein, vector specificity, ss/ds DNA ratio
AV2 13kd Unknown in group III, virus movement in groups I & II
BC1 34kd Virus movement, host ranges, and symptom development
BV1 30kd Virus movement and host ranges
49
Figure 1 Top: Symptoms associated with cotton leaf crumple virus (CLCrV). The typical
on cotton include downward curling of leaves with frequent vein distortion, vein
clearing, mosaic, floral distortion, and general stunting.
Bottom: Typical symptoms incited by cotton leaf curl virus (CLCuV). The
symptoms include upward curling of leaves. A marked swelling of leaf veins with
enhanced dark green coloration. In addition, leaf- like enations of various sizes can be
found on lower side of the leaf which originate in the leaf nectary. Infected plants are
stunted and distorted, bearing few flowers.
Figure 2. Schematic of genome organization of type members of geminivirus groups
CI JtepU.au.on
'" DNA aocumulaUoD.
DCI WO .... ~D.L;
drr-re::::.,:1.
~~-------~ CO
DK1 accYJUwaUoD
Figure 2
yz Coat prOLtlAl
"09II1II. •• 1.
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,. C_l pr.t.~ Wonm..a.l
51
Chapter Two
Molecular Characterization and Comparison of
Cotton Leaf Crumple and Cotton Leaf Curl Geminiviruses
ABSTRACT
52
Cotton leaf curl geminivirus (CLCuV) causes a serious cotton disease in Asia and
Africa. The symptoms of CLCu V are distinguishable from those of cotton leaf crumple
geminivirus (CLCrV) reported in North and Central Americas. Both viruses are whitefly
transmitted. However, the relationship between these two viruses is largely unknown.
Using a polymerase chain reaction (PCR) based technique, DNA A of both viruses was
amplified and cloned from total nucleic acids extracted from infected cotton leaves. The
DNA A component of each virus was amplified as two PCR fragments with two pairs of
oligonucleotide primers. These primers were designed according to the conserved regions
of the published, whitefly-transmitted geminivirus sequences. Electrophoretic analysis of
the PCR products indicated that the DNA A of CLCrV and CLCuV was approximately
2.6 kb and 2.7 kb, respectively. Southern blot analysis showed that CLCrV DNA and
CLCuV DNA did not hybridize with each other under high stringent conditions while they
hybridized slightly with each other under low stringent condition. These hybridization
data are consistent with partial sequence analysis of the coding regions of the ALl gene
and coat protein gene. Although both viruses share homology at the nucleic acid level,
CLCrV is more closely related to the new world geminiviruses such as bean dwarf mosaic
virus, tomato mottle virus, Abutilon mosaic virus, and potato yellow mosaic virus while
CLCu V is more closely related to the old-world viruses such as cassava latent virus,
tomato yellow leaf curl virus, and Indian cassava latent virus. Our data indicate that
CLCrV and CLCuV are two distinct and distantly related geminiviruses.
53
Introduction
Recent changes in the sweet potato whitefly (Bemisia tabaci Genn.) populations have
led to a marked increase in the incidence of two important viral diseases in cotton
(Gossypium hirsutum L.), cotton leaf crumple virus (CLCrV) and cotton leaf curl virus
(CLCuV). CLCrV was fIrst reported from California (Dickerson et al., 1954). Allen et
al. (1960) subsequently reported the disease in Arizona a few years later. CLCrV disease
is characterized by floral distortion, general stunting, foliar mosaic and downward curling
in cotton (Brown et aI1987). Losses resulting from CLCrV infection range from 21 to
86% depending on the age of plants at the time of infection (Allen et al., 1960, Brown et
ai, 1987, Van Schaik et ai, 1962). Host range of the virus includes numerous species
within the Malvaceae and Fabaceae families (Brown et ai, 1987). Monomers of 17-20 nm
and dimers of 17-20 x 30-32 nm were observed in CLCrV preparations partially purified
from infected red Kidney bean plants (Brown et ai, 1984).
CLCuV was fIrst reported in Africa (Nour & Nour, 1963). This virus can be
distinguished from CLCrV by symptoms on plants. Leaves of CLCuV infected cotton
plants curl upward instead of downward as do CLCrV infected leaves. In addition, vein
thickening and enations on the underside of the leaves are observed only on CLCu V
infected cotton. As with CLCrV infection, CLCuV-infected cotton bears very few flowers
and yield reduction varies with the age of plants at the initial infection. In Pakistan, some
cotton fields were wiped out entirely when the infection occurred early in the growing
season (personal observations). Geminivirus-like particles were found associated with the
diseased cotton plants (Khalid, S. personal communication). The virus can be transmitted
by B. tabaci in greenhouses from cotton to tobacco and cotton. Using polymerase chain
reaction (PCR) with geminivirus-specifIc oligonucleotide primers, Mansoor et al., (1993)
amplified a DNA fragment from CLCu V infected Nicotiana benthamiana plants. This
PCR fragment hybridized with an African cassava mosaic virus DNA probe, suggesting
CLCu V was a geminivirus.
54
Geminiviruses are a diverse and yet conserved virus group. Based on host ranges,
vector relationships, and number of genomic DNA, geminiviruses have been divided into
three subgroups (Francki et al., 1991). Subgroup III consists of all the whitefly
transmitted geminiviruses that infect dicotyledonous plants. Their genome consists of two
single-stranded DNA molecules, DNA A and DNA B. All the subgroup ill gemini viruses
sequenced so far have a similar genome organization (Lazarowitz et al., 1991) (Fig. 1).
Among fours genes on DNA A, ALl, AL2, and AL3 are involved in the viral DNA
replication and transcriptional regulation and ARI encodes the capsid protein. DNA B
carries two genes that affect disease symptoms and function in the virus intercellular and
systemic movement in infected plants. There is a considerable degree of homology at the
DNA level and the protein level among subgroup ill gemini viruses.
Both CLCrV and CLCuV infect dicotyledonous plants and are whitefly-transmitted.
Previous studies (Brown et al., 1984, Mansoor et al., 1993) suggested that they belong to
the subgroup III geminiviruses. Little infonnation is available on how these two cotton
infecting viruses are related to each other and with other subgroup ill gemini viruses. In
this paper, we report nucleic acid hybridization and sequencing analysis of the PCR
amplified viral DNA fragments of CLCrV and CLCuV DNA A components. Our study
indicates that CLCrV and CLCuV are two distinct and distantly related subgroup III
gemini viruses.
Materials and Methods
Virus isolates
The CLCrV isolate used in this study was originally collected in Arizona The virus
was maintained and propagated on greenhouse cotton plants.. The CLCu V isolate was
originally collected from cotton field in Multan, Pakistan and has been maintained on
cotton plants at the National Agricultural Research Center, Islamabad, Pakistan.
DNA extraction
55
A mini-DNA isolation technique was developed to extract total nucleic acids from
young, infected cotton leaves. This technique was modified from a procedure described
by Gawel and Jarret (Gawel et al., 1991). Briefly, infected leaves were harvested 24
hours before extraction. One hundred milligrams of cotton leaves were then pulverized in
liquid nitrogen, mixed with 20 volumes of extraction buffer (2% crAB, 20 mM EDTA,
1.4 M NaCI, 100 mM Tris-HCl, pH 8.0) preheated to 65°C. ~-mercaptoethanol was
added to a final concentration of 0.4%. The homogenized tissue extracts were incubated
at 65°C for 30 minutes, followed by two chloroform-isoamyl alcohol (24:1) extractions at
room temperature. Total nucleic acids in the aqueous phase were precipitated with the
addition of one volume of isopropanol. The final pellet was resuspended in TE buffer (10
mM Tris, pH 8.0, 1 mM EDTA) containing 50 jlg/ml RNase A.
peR primer design
Two pairs of degenerate oligonucleotide primers were designed for PCR amplification
ofCLCrV and CLCuV DNA A from the total cotton DNA. Sequences of six whitefly
transmitted, bipartite geminiviruses were aligned using the Pileup program in the
Wisconsin Sequence Software Analysis Package. Two highly conserved regions were
identified, one around nucleotide 500 in the coat protein gene and another around
nucleotide 1800 in the ALl gene of the geminivirus DNA A (Figs. 1 and 2). A forward
primer (F500 and F1800) identical to and a reverse primer (R500 and R1800)
complementary to the sequences in each conserved region were designed. F500 and RSOO
correspond to 20 nucleotides from 492 to 513 and F1800 and R1800 correspond to 22
nucleotides from 1797 to 1816 in the aligned multiple sequences. To accommodate non-
56
conserved nucleotides at certain positions, 4 folds of degeneracy were introduced to
primers F500 and RSOO, and 6 folds of degeneracy were introduced to primers FI800 and
RI800 (Fig. 2). Restriction sites XbaI (TCfAGA) and SstI (GAGCfC) were engineered
into the 5' termini of the reverse and forward primers, respectively, for the subsequent
cloning experiments. Four additional random nucleotides were added to the 5' of the
restriction sites to facilitate the restriction digestion by XbaI and Sst!. By using a
combination of F500 and RI800 primers and a combination of RSOO and F1800 primers,
the lower half and the upper half of the DNA A were amplified respectively (Fig. 1). All
the PCR and sequencing primers were synthesized by the DNA synthesis facility at the
Arizona Biotechnology Center.
PCR amplification of viral DNA
PCR reactions were carried out in 50 III of 10 mM Tris. HC1, pH 9.0, 50 mM KC1,
1.5 mM MgCh, 0.01 % gelatin, 0.1 % Triton X-lOO, 0.25 mM dNTP, 2.5 U Taq DNA
polymerase (Promega, WI), 0.4 11M each of a forward primer and a reverse primer, and
100 ng of total cotton DNA. The mixture was overlaid with 50 III mineral oil and
amplified by 40 cycles of reactions in a Temp-tronic thermocycler (Thermolyne, IL). Each
cycle consisted of denaturation at 94°C for 1 min, annealing at 57°C for 30 sec, and
polymerization at 72°C for 1.5 min. The polymerization step was incremented by 3
seconds after every cycle. PCR amplified DNA was analyzed by electrophoresis in a 0.9%
agarose gel.
Cloning of PCR fragments
Virus specific DNA fragments were excised from the agarose gel and recovered by
binding to, and elution from, a silica matrix (GeneClean, Bio 101, La Jolla, CA). CLCrV
DNA fragment amplified with primers F500 and R1800 were first digested with SstI and
XbaI enzymes. This DNA fragment was then ligated into pBS(+) plasmid (Stratagene, La
57
Jolla, CA) digested with the same enzymes to yield plasmid pCLCrVL. Because of an
internal XbaI site, the CLCrV DNA fragment amplified by RSOO and F1800 primers was
cloned by blunt-end ligation into the SmaI digested pBS(+) plasmid to yield plamid
pCLCrVU. For the cloning of CLCuV PCR DNA fragments, a TA cloning system
(Marchuk. et ai, 1991) was adapted. pBluescript SK(+) plasmids (Strategene, La Jolla,
CA) was first digested with EcoRV to yield blunt ends. An additional dT residue was
added to the 3' end of the blunted DNA by incubation with Taq DNA polymerase in the
presence of 2 mM dTTP for 2 hours at 720 C. One JlI of the PCR product was directly
ligated into the EcoRV digested, Taq DNA polymerase modified vector. The plasmids
containing the CLCuV DNA fragments amplified with primers F500 and R1800 and with
primer RSOO and F1800 were designated as pCLCuVL and pCLCuVU, respectively.
Unless stated otherwise, standard DNA manipulation techniques as described by
Sambrook et al. (1989) were followed.
Southern hybridization
An enhanced chemilluminescent (ECL, Amersham, Arlington Heights, IL) technique
was used in Southern hybridization analysis. Plasmids containing cotton gemini virus DNA
fragments were purified using a PEG technique (Sambrook et ai, 1989). Following
digestion with appropriate restriction enzymes, viral DNA inserts were fractionated by
agarose gel electrophoresis and subsequently transferred onto Nylon membrane (Sigma,
St. Louis, MO) using a semidry transfer apparatus (Biorad, Richmond, CA). Briefly, the
agarose gel and membrane was sandwiched between two six-sheet Whatman
chromatography papers saturated with 0.5 X TBE buffer. An electric current of 2
rnA/cm2 was applied for 1 hour to complete the transfer. Probes specific for CLCrV and
CLCu V were labeled in PCR reactions containing an equal molar ratio of dTTP and
Fluorescence(Fl)-dUTP (Amersham, Arlington Heights, IL) in addition to other three
58
deoxyribonucleotides. Prehybridization, hybridization with Fl-dUTP labeled probes, and
the subsequent detection by chemilluminescence were carried out as described by the
manufacturer.
Nucleic Acid Sequencing
Nucleotide sequences were determined by dideoxy-nucleotide chain termination
reactions (Sanger et al., 1977) with Sequenase II (United State Biochemical, Cleveland,
OH) according to manufacturers instructions. Purified double-stranded DNA containing
cotton geminivirus inserts was denatured and used directly as templates for sequencing.
Partial sequences of the coat protein gene and the ALl gene of the cloned DNA were
obtained by priming with T3 and 17 primers.
Sequence analysis
Partial sequence fragments of CLCrV and CLCuV were assembled with the
GELASSEMBLE program in the Genetic Computer Group Software package. The
assembled sequences were compared with each other, and with the available geminivirus
sequences in the Genbank and EMBL databases with FASTA program (Pearson et al.,
1988) available in the same software package.
Results
Total cotton DNA extraction and peR amplification of viral DNA
Cotton contains high level of phenolic terpenoid compounds and tannins (Katterman et
al., 1983) that complicate DNA extraction. We developed a simple yet effective cotton
DNA extraction procedure that overcame that complication. This procedure consistently
yielded high quality total DNA preparations. Purified DNA migrated as a single, sharp
DNA band of high molecular weight when analyzed by agarose gel electrophoresis (Fig.
3, lane 9). The critical factor in the successful extraction of cotton DNA was the high
buffer:tissue ratio, ususally 20:1. Smaller buffer:tissue ratios resulted in significant
reduction in both the quality and quantity of the DNA obtained (data not shown).
59
The effectiveness of the extracted DNA as PCR templates was detennined with both
primer pairs as outlined in Fig. 2. A single DNA fragment was amplified from each
primer pair from the CLCrV -infected cotton DNA (Fig. 3) lanes 2 and 6. No DNA
products were amplified from DNA sample extracted from healthy cotton Oanes 3 and 7)
or from control with no added exogenous DNA templates Oanes 4 and 8). This result
indicated that the designed primers were specific for geminiviruses and the observed DNA
fragments were amplified from CLCrV DNA. Total DNA preparations extracted with the
procedure described in this report were excellent templates for PCR reactions.
Size comparison of PCR amplified CLCrV and CLCu V DNA
Total DNA extracted from both CLCrV and CLCuV-infected cotton leaves was used
as PCR templates. Viral DNA fragments amplified by PCR were analyzed by agarose gel
electrophoresis (Fig. 4). DNA fragments of approximately 1.2 kb and 1.4 kb were
detected from CLCrV-infected cotton DNA amplified with the primer pair F500 and
R1800 (lane 4) and with the primer pair R500 and F1800 (lane 2), respectively. A
fragments of approximately 1.2 kb and a slightly larger fragment of 1.5 kb were detected
from CLCuV-infected cotton DNA amplified with the same two pairs of primers Oanes 3
and 1). Based on the agarose gel electrophoresis data, the total length of CLCrV DNA A
was calculated as about 2.6 kb while that of CLCuV DNA A was about 2.7 kb. The
difference in the size of DNA A was sufficiently large enough to suggest that CLCrV and
CLCu V are two different geminiviruses.
Differential hybridization of CLCrV and CLCuV DNA A components
To further differentiate these two viruses, four probes labeled with FJ-dUTP were
synthesized by PCR. The labeling reactions were carried out with the same two pairs of
60
primers and cotton DNA from infected plants as templates. These probes corresponded to
the upper and lower fragments amplified from the two viruses. Plasmids pCLCrVL and
pCLCrVU, containing the lower portion and upper portion of CLCrV DNA A, and
plasmids pCLCu VL and pCLCu VU, containing the lower portion and upper portion of
CLCuV DNA A, were used for the hybridization. Viral DNA inserts were released by
restriction digestion with SstI and HindIII in pCLCrVU and pCLCuVU, with SstI and
XbaI in pCLCrVL, and with SstI in pCLCuVL. The digested DNA fragments were
fractionated on a 0.9% agarose gel (Fig. 5A), transfered to Nylon membranes, and
hybridized with Fl-dUTP labeled homologous and heterologous probes. The hybridized
Nylon membranes were subsequently washed under two conditions. The low stringent
washing consisted of two 15-minute agitation in IX SSC containing 0.1 % SDS at room
temperature, followed by two 15-minute washes in 0.5 X SSC containing 0.1 % SDS at
650 C. In the high stringent washing, the last two washes were carried out in O.IX SSC
containing 0.1 % SDS.
Under the low stringent condition, each probe hybridized strongly with its own DNA
fragments (Fig. 5B). Probes representing the lower portion of both CLCrV and CLCuV
also hybridized with the equivalent DNA fragment of the other virus while probes
representing the upper portion of the DNA A components did not Under the high
stringent condition, each probe hybridized only with its own DNA. Little hybridization
signal was detected with heterologous DNA fragments (Fig. 5C). These data further
suggest that the two cotton geminiviruses are distinct and yet distantly related viruses.
The upper portion of the DNA A (Fig. 1) contains a common intergenic region which is
highly conserved within the two DNA components of a virus and yet quite diverged
between geminiviruses (Lazarowitz, 1992). On contrary, the lower portion of the DNA A
contains mostly coding regions with little intergenic region. Coding regions on the DNA
61
A of the subgroup ill geminivirus are known to share considerable sequence homology at
the nucleic acid level. The absence of the intergenic region probably explains the
preferential cross hybridization of the DNA fragments representing the lower portion of
theDNAA.
Analysis of partial DNA A sequences of CLCrV and CLeu V
Partial sequences were determined from the coat protein gene and ALl gene region of
CLCrV and CLCuV from their respective DNA clones (Fig. 6). Equal number of
nucleotides were used to analyze the sequences of the capsid protein gene and the ALl
gene regions from both viruses. Nucleotide sequences of CLCrV and CLCuV were
aligned with each other using the FASTA program (Pearson and Lipman, 1988). The
capsid protein regions showed the highest homology of 74% identity between CLCrV and
CLCuV (Fig. 6A), whereas the similarity in ALl regions was about 67% (Fig. 6B).
In order to determine how different these two geminiviruses are from each other in
relation to other geminiviruses, partial sequences of ALl gene and coat protein gene of
CLCrV and CLCuV were compared to all geminiviruses available in GenBank and EMBL
databases. The compiled results of FASTA search are shown in Tables 1 and 2.
Similarity scores between two compared sequences were calculated as described by
Pearson and Lipman (Pearson et a/., 1988). Higher scores indicate more closely related
viruses. All the geminiviruses list in the tables were sorted according to the similarity
scores in the ALl gene region. When CLCrV sequences were used as query sequences,
Abutilon mosaic virus (AbMV), tomato mottle virus (ToMo V), bean dwarf mosaic virus
(BDMV), and potato yellow mosaic virus (PYMV) produced highest scores (Table 1).
All these viruses were classified as the 'New World' geminiviruses (Howarth &
Vandemark,1989). On the contrary, when CLCuV sequences were used as query
sequences, cassava latent virus (CL V), Mrican cassava mosaic virus (ACMV), tomato
yellow leaf curl virus (fYLCV) and Indian cassava mosaic virus (lCMV) produced the
highest scores. All of them were classified as the 'Old-World viruses (Howarth &
Vandemark,1989). These data suggest that CLCuV is more closely related to
geminiviruses of the old world and CLCrV to the geminivirus in the new world. The
FASTA scores in reciprocal tests between CLCrV and CLCuV are the lowest among all
the group ill geminiviruses , indicating that they are the least related among the
geminiviruses compared. Analysis of the partial viral sequences confmned our earlier
observations that CLCrV and CLCuV are distantly related geminiviruses.
Discussion
62
Mansoor et af. (1993) recently reported amplification of CLCuV DNA from
experimentally infected N. benthamiana plants. However, they failed to extract and
amplify CLCuV DNA from infected cotton plants. We have reported here for the first
time the amplification of both CLCrV and CLCuV DNA A directly from DNA
preparations extracted from infected cotton plants, the natural host for both viruses.
Furthermore, we have shown, by analyzing PCR amplified DNA fragments and partial
DNA sequences, that CLCrV and CLCuV were two distinct and distantly related
geminiviruses. CLCrV and CLCu V are the only two geminiviruses that infect cotton and
the only virus diseases of importance of this crop (Watkins, 1981).
Comparisons between these two viruses and with other geminiviruses indicate that the
capsid protein gene region is more conserved than the ALl gene region (Fig. 5, Table 1).
A similar trend was observed in other studies (Dry et al., 1993, Gilbertson et al., 1993,
Lazarowitz et al., 1992). The higher degree of conservation in the capsid protein region is
probably due to their transmission through a common vector (Lazarowitz et al., 1992). In
Table 1, all the viruses that showed high degree of homology in the capsid protein region
were whitefly transmitted. Beet curly top virus (BCTV), a leafhopper transmitted,
63
dicotyledon geminivirus, also has considerable homology with both CLCrV and CLCuV in
the ALl gene region, but not in the capsid protein region. This observation is consistent
with other reports (Faria et al., 1994, Gilbertson et al., 1993, Howarth et al., 1989). Our
sequence analysis shows that CLCrV is more closely related to the new world
geminiviruses such as AbMV, ToMoV, BDMV, and PYMV, and CLCuV is more closely
related to the old world geminiviruses such as CLV ,ACMV, TYLCV, and ICMV, as
classified by Howarth & VandeMark (1989). According to their hypothesis, CLCuV is
likely to have originated in the old world and CLCrV in North America. Even though
only about 350 nucleotides each from the ALl gene and the CP gene were used in our
sequence analysis, the resulting rankings of the related geminiviruses clearly fall into
clusters as defined in other studies (Faria et al., 1994, Howarth et al., 1989). Thus, these
short sequence fragments are adequate to determine the relationship between CLCrV and
CLCuV.
The key to our successful PCR amplification of viral DNA from total cotton DNA was
the high quality of the DNA preparation. We experimented with several different
procedures for total DNA extraction, which produced DNA preparations of either low
yield or poor qUality. The DNA extraction procedures described by Katterman and
Shattuck (Katterman et al., 1983) was quite complicated and required purification of
cotton nuclei before the actual DNA extraction. Their method has consistently resulted in
low DNA yield in our hands. A DNA extraction procedures reported by Hughes & Galau
(Huges et al., 1988) also did not produce satisfactory results. They used low temperature
to avoid phenolic and other organic denaturants in initial steps and SDS and potassium
acetate precipitation to remove proteinaceous materials. A procedure similar to the one
we described but with an extraction buffer containing 1M Ches, pH 9.1,0.5 M EDTA, 4M
NaCl, 0.5% NP-40, 10% SDS, and 0.2% b-mercaptoethanol did not work very well. The
64
procedure described in this report consistently produced high quality DNA preparations.
An advantage of this DNA extraction procedure is the short processing time and the small
starting material required. The entire procedure can be accomplished less than one hour
while some of the other procedures take more than one day. This saving in time and in
materials will expedite large scale field survey of cotton geminiviruses by PCR.
PCR primers used in this study were designed according to the two conserved regions
of the whitefly-transmitted subgroup m geminiviruses. They were capable of priming the
amplification of DNA A from two very distantly related group m geminiviruses. We did
not observe any non-specific band amplified from either virus infected or healthy DNA
preparations (Fig 2). Based on their performance in these experiments, these primers
should be capable of detecting any other whitefly transmitted group III gemini viruses
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Stanley, J. and Gay, M.R. 1983. Nucleotide sequence of cassava latent virus DNA. Nature 301, 260-262
Stanley,J., Markham,P.G., Callis,R.J. and Pinner,M.S. 1986. The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus. EMBO J. 5, 1761-1767.
Torres-Pacheco, I., Garzon-Tiznado, 1. A., Jimenez, B., Herrera-Estrella, L. and RiveraBustamante, R. F. 1993. Complete nucleotide sequence of pepper huasteco virus: analysis and comparison with bipartite geminiviruses. J. Gen. Virol. 74,2225-2231.
van Schaik, P. H., Erin, D. C., and Garber, M. 1. 1962. Effects of time on symptom expression of the leaf-crumple virus on yield and quality of fiber in cotton. Crop. Sci. 2,275-277.
68
Table 1. Comparison ofCLCuV and CLCrV Partial Sequences in ALl and CP Regions With
Other Geminiviruses
A. CLCrV B. CLCuV
Virus ALI Gene CPGene Virus ALI Gene CPGene
CLCrV 1036 1276 CLCuV 1016 1276 AbMV 776 919 CLV 677 633 ToMoV 755 891 ACMV 670 598 BDMV 731 933 TYLCV 670 610 PYMV 717 898 ICMV 628 821 SLCV 654 877 TLCV 593 711 BGMV 622 884 SLCV 512 716 TGMV 608 912 BDMV 499 702 CLV 528 603 TGMV 499 695 ACMV 521 638 PYMV 498 695 BCTV 518 AbMV 470 688 ICMV 503 639 PHV 464 697 TLCV 497 463 ToMoV 463 662 TYLCV 461 612 BGMV 457 744 PHV 455 802 CLCrV 421 639 CLCuV 421 639 BCTV 325
Note: The capsid protein of BCTV did not show any homology with either CLCrV or CLCu V when analyzed with FASTA program.
Abbreviations for geminiviruses and their references: AbMV= Abutilon mosaic virus (Frischmuth et al .• 1990), ACMV= African cassava mosaic virus (Morris et al .• 1990), BCTV=beet curly top virus (Stanley et al .• 1986), BDMV=bean dwarf mosaic virus (Hidayat et al .• 1992), BGMV= bean golden mosaic virus (Howarth et al .• 1985), CLV= cassava latent virus (Stanley et al .• 1983), ICMV=indian cassava mosaic virus (Hong et ai, 1993), PHV=pepper huasteco virus (Torres-Pacheco et al .• 1993), PYMV=potato yellow mosaic virus (Coutts et al .• 1991), SLCV = squash leaf curl virus (Lazarowitz et al .• 1991), TGMV= tomato golden mosaic virus (Hamilton et al .• 1984), TLCV= Tomato leaf curl virus (Dry et al .• 1993) , ToMoV=tomato mottle virus (Abouzid et al .• 1992), TYLCV=tomato yellow leaf curl virus (Navot et al .• 1991).
69
Figure. 1. A generic genomic map of DNA A and locations of PCR primers and
schematic representation of DNA fragments amplified by PCR. Solid arrows represent
open reading frames and direction of transcriptions conserved among subgroup III
geminiviruses. Notice that there is a larger intergenic region in the upper half of the DNA
A. Primers RSOO and F1800 are used to amplify the upper half of the DNA A. Primers
F500 and R1800 were used to amplify the lower half of the DNA A.
Figure. 2. Alignment of the DNA sequences of the six bipartite ,whitefly transmitted
subgroup ill gemini viruses using the Pileup program in the Wisconsin Sequence Software
Analysis Package. Conserved sequences are denoted in bold. Two pairs of degenerate
oligonucleotide primers are designed for PCR amplification of geminivirus DNA A. A
forward primer (F500 and F1800) and a reverse primer (RSOO and R1800) are designed at
each conserved region. Restriction sites XbaI (fCT AGA) and SstI (GAGCTC)
engineered to the 5' termini of the primers are denoted in italics. Four additional random
nucleotides were added to the 5' of the restriction sites in each primer. Degenerated
nucleotides are denoted as: Y=C,T; R=G,A; W=T,A; B=C,G,T; V=A,C,G.
Abbreviations: ACMV= African cassava mosaic virus (Morris et al. , 1990), ABMV=
Abutilon mosaic virus (Frischmuth et ai, 1990), BGMV= bean golden mosaic virus
(Howarth et al. , 1989), CLV= cassava latent virus (Stanley et al. , 1983), SLCV = squash
leaf curl virus (Lazarowitz et al. , 1991), TGMV= tomato golden mosaic virus (Hamilton
et al., 1984).
Figure 3. Agarose gel electrophoresis of PCR amplified DNA fragments from cotton leaf
crumple virus (CLCrV) DNA A. Each lane represents DNA products from 10 ng of total
cotton DNA amplified for 40 cycles. BRL 1 Kb standard was used to calculate the length
of PCR products. Total DNA preparations extracted from healthy (lanes 3 and 7) and
CLCrV -infected (lanes 2 and 6) cotton were amplified with either primer pair F500 and
70
R1800 (lanes 2, 3and 4) or primer pair RSOO and F1800 (lanes 6,7 and 8). As controls,
PCR reactions with no DNA templates were carried out with the primer pair F500 and
R1800 (lane 4) and the primer pair RSOO and F1800 (lane 8). Lane 9 shows total cotton
DNA extracted as described in material and methods. PCR amplified and total DNA was
analyzed by electrophoresis in a 0.8% agarose gel. Note that distinct DNA fragments
were amplified only from CLCrV -infected DNA samples (lanes 2 and 6). No DNA
fragments were amplified from healthy cotton DNA
Figure. 4. Comparison of PCR amplified DNA A fragments of cotton leaf crumple virus
(CLCrV) and cotton leaf curl virus (CLCuV). BRL 1 Kb standard; was to calculate the
size of PCR products, Lanes 1) Upper portion of CLCuV DNA A and Lanes 2) upper
portion of CLCrV DNA A were amplified with primers RSOO and F1800. Lanes 3) Lower
portion of CLCuV DNA A and Lanes 4) lower portion of CLCrV DNA A amplified with
primers F500 and R1800. Notice that CLCuV DNA fragments are slightly larger than the
frgaments of CLCrV DNA
Figure. 5. Enchanced chemilluminscent Southern hybridization of cotton leaf crumple
virus (CLCrV) and cotton leaf curl virus (CLCuV) DNA A with reciprocal probes.
Plasmids pCLCrVL, pCLCrVU, pCLCuVL, and pCLCuVU were digested with restriction
enzymes to release the viral DNA inserts. Gel fractionated DNA fragements (A) were
transferred to Nylon membranes. Fluorescence-dUTP labeled probes specific for each
cloned insert were synthesized by PCR. Four identical sets of blots were prepared. Each
blot was hybridized with a different Fl-dUTP labeled probe at either a low stringent
condition (B) or a high stringent condition. Sources of viral DNA are indicated on the top
of each lane. Hybridization probes are indicated on top of each panel. Note that DNA
fragements representing the lower portion of DNA A from either CLCrV (CLCrV -L) or
CLCuV (CLCuV-L) cross-hybridized with each other at the low stringent condition (B)
but not at the high stringent condition.
71
Figure. 6. Alignment of the partial DNA sequences of the capsid protein gene region
(A) and ALl gene region (B) of cotton leaf crumple virus (CLCrV) and cotton leaf curl
virus (CLCuV). Sequences were determined from cloned PCR DNA fragments of each
virus. Sequences aliognments were generated with the FASTA program (pearson and
Lipman, 1988) in the Wisconsin Genetic Computer Groups softer package with all
parameters at the default settings. Identical nuc1eotides are indicated by vertical lines.
Gaps were inserted to allow maximum alignment and denoted as dash.
73
A. 481 530
ACMV GGCCTGGAGG AACAGGCCCA TGTA~GAAA GCCCATGATG TACAGGATGT CLV GGCCTGGGTG AACAGGCCCA TGTA~AGAAA GCCCACGATG TACAGGATGT SLCV TGCATGGGTT AACAGGCCCA TGTA~~AA GCCCAGGATC TATCGCACAA AbMV TGAATGGGTG CACAGGCCCA TGTA~A~AA GCCCAGGATC TACCGGACGC BGMV TGCATGGGTC AACAGGCCCA TGTA~AGAAA GCCAAGGATA TATCGGATGT TGMV TGCTTGGGTT AACAGGCCCA TGTA~A~AA GCCCGAGATA TATCGATCAC
I:5..Q.Q 5' CCGGGAGCTCACAGGCCCA TGTAYAGRAA GCC 3' .RS.Q.Q. 3 ' TGTCCGGGT ACAT,BTCXTT CGGAGATCTAACG 5'
B.
1781 1830 ACMV GAAGATAGTG GGAATGCCAC CTTTAATTTG AAC~TTTC CCGTATTTCG CLV GAAGATAGTG GGAATTCCAC CTTTAATTTG AAC~TTTC CCGTATTTCG SLCV CACGATTGAT GGGATTCCAC CTTTAATTTG AAC~GGCTTT CCATATTTAC AbMV CACGATTGCT CGGATTCCTC CTTTAATTTG AAC~GGCTTG GCTAACTTGC BGMV CACGATTGAT GGTATTCCTC CTTTAATTTG AAC~GGCTTT CCATATTTAC TGMV CACGATTGAC GGGATACCTC CTTTAATTTG AACTGGCTTT CCGTATTTAC
P1800 R1800
5' CCGGGAGCTCCCWC CTTTAAT'l'TG AACBGG 3' 3 ' GGWG GAAATTAAAC TTGVCCAGATCTAACG 5 '
Figure. 2
()
I CLCrV-L n CLCuV-L ~ CLCrV-U ~ CLCuV-U I'"
:n CLCrV-L n
I CLCuV-L (; <C ~ C CLCrV-U • I'" -or CLCuV-U CD (J1
CLCV-L n CLCurV-L ~ • CLCV-U <: CLCuV-U C
CLCrV-L n CLCuV-L ~
I CLCrV-U ~ CLCuV-U
OJ
, CLCrV-L n CLCuV-L ~ CLCrV-U ~ CLCuV-U I'" , CLCrV-L n .' CLCuV-L (;
~ CLCrV-U l-CLCuV-U
CLCrV-L n
• CLCuV-L ~ CLCrV-U ~ CLCuV-U
CLCrV-L n CLCuV-L P
• CLCrV-U ~ CLCuV-U
»
. j-
_ CLCrV-L
, CLCuV-L . .' ~
CLCrV-U
_CLCuV-U
-..l VI
A CLCrV ACAGGCCCATGTACAGGAAGCCCAGAATCTATCGCACGTTAAGAACGCCTGACATTCCGA
111111111111111111111111111111111 I II 1111 II II 1111 I CLCuV ACAGGCCCATGTACAGGAAGCCCAGAATGTATCGGATGTACAGAAGTCCGGATGTTCCAA
CLCrV GAGGCTGTGAAGGGCCATGTAAAGTCCAGTCATATGAACAGCGCCACGATGTCTCACACG II 11111 III 11111111 II II II I III I I II 111111 II
CLCuV AGGGTTGTGAAGGCCCATGTAAGGTACAATCTTTTGAGTCGAGACATGATGTCGTTCATA
CLCrV TGGGTAAGGTAATGTGTATATCTGATGTCACACGTGGTAATGGTATCTCCCTCCGTGTCG I 11111111111111111 11111111 II 111111 III I III III I I
CLCuV TTGGTAAGGTAATGTGTATTTCTGATGTTACTCGTGGTGTCGGTTTGACCCATCGTATTG
CLCrV GTAAGCGTTTCTGTGTTAAGTCTGTATATATTTTAGGGAAAATATGGATGGATGAGAATA 1111 11111 11111 11111 II III 1111111 II 11111111111111 1111
CLCuV GTAAACGTTTTTGTGTCAAGTCAGTTTATGTTTTAGGTAAGATATGGATGGATGAAAATA
CLCrV TTAAGCTGAAGAACCACACGAATAGCGTCATTTTTTGGCTGGTTAGAGATCGTAGACCAT I III 11111 111111111 II II II I I II 111111 I II
CLCuV TAAAGACCAAGAATCACACGAATTCGGTGATGTTCTTTTTAGTCCGCGATCGACGTCCTG
CLCrV ATGGCACTCCTATGGACTT II II III III II
CLCuV TTGACAAACCTCAGGATTT
B. CLCrV CCACCTTTAATTTGAACCGGTTTCCCGTACTTGCAATTTGACTGCCAGTCTTTTTGGGCC
II 11111111111111 II III 11111111 II II 11111111 1111 II CLCuV CCTCCTTTAATTTGAACGGGCTTCGCCGTACTTTGTGTTGGATTGCCAGTCCCTTTGTGC
CLCrV CCCAAGAAGCTCTTTCCAATGCTTTAGCTTTAGGTATTGCGGTGCTACGTCATCAATGAC III I 1111111 I I I II III 111111 I 11111111111111
CLCuV CCCCATGAACTCTTTAAAGT------GTTTGAGGAAATGCGGGTCGACGTCATCAATGAC
CLCrV GTTATAGGCAACATTATTCGAGTACACTCGATTATTGAAGTCCAGGTGTCCACTAAGGTA 11111 I I II III II I I I 111111 1111111 I II
CLCuV ATTATACCAGGCGTCGTTACTGTAGACCTTGGGACTCAGGTCCAGATGTCCACACAAATA
CLCrV ATTATGTGGGCCTAAAGCACGAGCCCACATCGTCTTGCCTGTTCTAGAATCACCTTCCAC 11111111 II II I 1111111111111111 II II I I III 11111 I
CLCuV GTTATGTGGTCCCAATGACCTAGCCCACATCGTCTTCCCCGTACGACTATCTCCTTCAAT
CLCrV TATGAGACTAACTGGTCTTA
I III 11111 CLCuV CACTATACTTTGAGGTCTGT
76
77
Chapter Three
Genome Organization and Sequence Analysis Of Cotton Leaf Crumple Geminivirus DNA A
ABSTRACT
The complete genomic DNA A of the whitefly-transmitted cotton leaf crumple virus
(CLCrV) has been cloned and sequenced. CLCrV DNA A consists of 2630 nucleotides.
Sequence analysis indicates that the CLCrV DNA A has a genome organization similar to
subgroup III geminiviruses with five open reading frames (ORFs) (A VI, ACl, AC2, AC3,
AC4). Two unexpected ORFs were identified in CLCrV DNA A component. The ORF on
the viral sense strand, termed as A V2 was found completely inside but in the complementary
strand to ACI ORF. The ORF on the complementary strand termed as AC5, is contained
within ORF AVI in the opposite direction. Comparisons of the nucleic acid sequences and
amino acid sequences showed that CLCrV (A VI) shares highest degree of similarity with
that of Abutilon mosaic virus, bean dwarf mosaic virus (BDMV), and potato yellow mosaic
virus (pYMV). The AC 1 gene of CLCrV show high degree of similarity with those of
squash leaf curl, tomato mottle virus, PYMV, BDMV and other group III geminiviruses from
the western hemisphere.
Introduction
Cotton leaf crumple geminivirus CCLCrV) is one of the few viruses infecting cotton
(Gossypium hirsutum L.). CLCrV causes floral distortion, mosaic and general stunting
and foliar malformation in cotton (Brown et al., 1987). This virus was first reported in
California (Dickson et ai, 1954). Allen et at. (1960) subsequently reported the disease in
Arizona a few years later. CLCrV -like disease was also reported in India in 1977 (Mali,
1977).
78
Yield losses from CLCrV infection ranging from 35% to 81 % were observed,
depending on the age of the plants when infected (Allen et al., 1960; van Shaik et al.,
1962; Butler et al., 1986; Brown et al., 1987). The virus is transmitted by the sweet
potato whitefly (Bemisia tabaci Genn.) and its host range includes numerous species
within the Malvaceae and Leguminosae (Brown and Nelson, 1987). Monomers of 17-20
nm and dimers of 17-20 X 30-32 nm were observed in CLCrV preparations partially
purified from infected Red Kidney bean plants (Brown and Nelson, 1984).
Geminiviruses are characterized by their small (20x30 nm) twinned isometric particles
and circular ssDNA genomes of about 2.5 to 2.8 kb. Member of the geminivirus group
can be divided into three distinct subgroups based on the number of genomic DNA, their
insect vector and the types of plants they infect (Lazarowitz, 1992). The viruses in
subgroup I contain a single DNA component and infect monocotyledonous plants. They
are exclusively transmitted by leafhoppers. Subgroup II consists of viruses which are also
transmitted by leafhoppers and have single genomic DNA. Viruses in this subgroup infect
several species of dicotyledonous plants. Subgroup III consist of viruses with a bipartite
genome (designated DNA A and B) which infect dicotyledonous plants and are
transmitted by whiteflies.
Although the transmission, morphology, host range, symptomatology, and economic
impact have been extensively studied, little is known about the molecular biology of
CLCrV and its relationship with other whitefly-transmitted geminiviruses. Based on the
whitefly transmission and the host range within dicotylednous families, CLCrV appears to
be a member of subgroup III geminivirus. In this communication, we report the complete
nucleotide sequence and the genome organization of the CLCrV DNA A, and its
relationship with other whitefly transmitted geminiviruses. Sequence analysis confmns
that CLCrV is a member of subgroup ill geminivirus.
Materials and Methods
Virus isolate and cloned DNAs
79
The CLCrV isolate used for cloning was originally obtained in Arizona. The virus was
maintained and propagated on greenhouse cotton plants. The plasmids containing the
CLCrV DNA fragments, pCLCrVL and pCLCrVU were described in Chapter Two.
Another clone pCLCrVIlO was generated using the primers designed based on the
sequences in the intergenic region of the CLCrV. A reverse primer RIlO
(T ATGGTGGGCCCAGGAGAG1) complementary to nucleotides 90 to 110 and a
forward primer F200 (GGAGCCCCCI"I"l"l I GTCGTT A) corresponding to nucleotides
200 to 220 for F200 were synthesized. Restriction sites SstI (GAGCTC) and Pst!
(CTGCAG) were engineered into the 5' termini of the reverse and forward primers,
respectively, for the subsequent cloning experiments. Four additional random nucleotides
were added to the 5' end of the restriction sites to facilitate the restriction digestion by
PstI and SstI. PCR reactions and cloning were carried out as described in Chapter 2.
Unless stated otherwise, standard DNA manipulation techniques as described by
Sambrook et al. (1989) were followed. A set of nested, overlapping subclones were
generated by exonuclease III digestion (Henikoff, 1984) for each clone. Subclones of
approximately 150 .... 200 nucleotides apart were selected for sequencing.
Nucleotide sequence determination
The complete nucleotide sequence of the DNA A was determined in both directions with
Sequenase II (United States Biochemical, Cleveland, OH) according to the manufacturer's
instructions and with an Applied Biosystems Automated DNA Sequencer Model 373A in
the dideoxy-nucleotide chain termination reactions (Sanger et aI., 1977), using either T3 or
80
17 oligonucleotides as primers. Both DNA strands were sequenced to ensure accuracy.
Compressions in some sequences were resolved by replacing dGTP with dlTP in sequencing
reaction.
Sequence analysis
The total sequence of the viral DNA A was assembled with the GELASSEMBLE
program in the Genetic Computer Group (GCG) Software package. The assembled
sequences were initially compared with the available gemini virus sequences in the Genbank
and EMBL databases with FASTA program (pearson & Lippman, 1988) available in the
same software package. Determination of nucleotide identity and amino acid similarity
among bipartite geminiviruses sequences was performed using GCG GAP program. A gap
weight of 5.0 and a gap length weight of 0.3 were used for calculating nucleotide identity
and a gap weight of 3.0 and gap length of 0.1 were used for amino acid comparisons.
Relationship between the conserved domains of different geminiviruses were analyzed using
PILEUP program of GCG package. Possible functional domains were identified using
MOTIF program of the same package.
Results and Discussion
Nucleotide sequence and genome organization of CLCrV DNA A
Nucleotide sequences of clones pCLCuVL, pCLCuVU were determined from both
strands. Sequences derived from pCLCuVL and pCLCuVU corresponded to nucleotides
474 to 1760 and nucleotides 1760 to 474, respectively. These two clones overalped with
each other by 22 nucleotides at one end and 20 nucleotides at the other end. In order to
verify sequence accuracy and fidelity of the PCR cloning, an independent clone
pCLCrV110 was generated using the primer set described in the materials and methods.
This clone overlaps the entire CLCrV but 100 nt ofpCLCrVU. Sequence determined
from the two strands of the clone were identical to the sequences derived from the other
81
two clones, indicating the high fidelity of the PCR cloning and the accuracy of the
sequencing reaction. The first nucleotide in intergenic region is designated as nucleotide
one. The length of CLCrV DNA A was determined to be 2630 nucleotides. Complete
nucleotide sequence of the virion strand of CLCrV DNA A is shown in Fig. 1. Based on
computer analysis and comparison with other geminiviruses, a total of seven open reading
frames in both the viral and the complementary strands with the potential to encode
proteins of Mr greater than 10 K were predicted (Table 1). The genome organization
derived from the sequence is schematically represented in Fig. 2. The nucleotide
sequence and the genomic organization indicate that CLCrV is a typical bipartite, whitefly
transmitted geminivirus. The virion strand of CLCrV DNA A contains two ORFs capable
of encoding products of 29.2 kd (CP) and 11.8 kd (A V2). CP ORF is similar in amino
acid sequence and genome position to capsid proteins of other whitefly vectored
geminiviruses. The complementary strand is predicted to encode 5 ORFs. The ORFs
were designated as ACt (40.1K), AC2 (15.9K), AC3 (15.7K), AC4 (14.8K) and AC5
(10.2K) based on size, position and sequence similarities with ORFs of other
geminiviruses (Fig. 2, Tables 1 and 2). CP, AC1, AC2 and AC3 encoded on CLCrV
DNA A are well conserved in other geminiviruses (Lazarowitz, 1992). AC4 ORF was
reported in a few geminiviruses including tomato golden mosaic virus (TGMV) African
cassava mosaic virus (ACMV), beet curly top virus (BCTV) and tomato leaf curl virus
(TLCV). Two ORFs, one on viral strand (A V2) and other on complementary strand
(AC5) are not well characterized. The AC5 ORF has previously been reported in pepper
huasteco virus (PHV) (Pacheco et al., 1993). The Tblastn search revealed that this ORF
is also present in some other viruses (data not shown). Since AC5 ORF is encoded on the
opposite orientation and entirely within the highly conserved CP ORF, it is not clear
whether there is a functional AC5 gene in gemini viruses. There is no report whether or
82
not AV2 exists in other whitefly transmitted geminiviruses (WTGs). In comparison with
other WTGs, the organization of CLCrV DNA A is similar to, with the exception of one
additional ORF each in the viral strand and in the complementary strand
Comparison of entire nucleotide sequence
Entire nucleotide sequence of CLCrV was compared with 16 whitefly geminiviruses
infecting dicots using GAP program of GCG package. The percent similarity is shown in
Table 2. The geminiviruses used in this comparison are grouped on the basis of their
geographic location, as the 'Old World', and the 'New World' geminiviruses (Howarth &
Vandemark, 1989; Abouzid et al., 1992). CLCrV showed greatest degree of identity with
bean dwarf mosaic virus (BDMV) (74%) (Hidayat et al., 1992), followed by squash leaf
curl virus (SLCV) (73%) (Lazarowitz et al., 1991), potato yellow mosaic virus (PYMV)
(73%) (Coutts et al., 1991), and other geminiviruses from the 'New World' whereas it
showed least homology with geminiviruses from 'Old World' such as cotton leaf curl
(CLCuV) (59%) (Chapter 4) followed by tomato yellow leaf curl (fYLCV) (60%) (Navot
et al., 1991), cassava latent virus (CL V) (60%) (Stanley et al., 1983), and Indian cassava
mosaic virus (ICMV) (60%) (Hong et al., 1993). Pepper huasteco virus (PHV) (Torres
Pacheco et al., 1993), a 'New World' virus has 65% identity with CLCrV, a score
between the 'Old World' geminiviruses and 'New World' geminiviruses. This observation
is consistent with previous studies that viruses from Western Hemisphere show close
relationship with each other but are distantly related with geminiviruses of the Eastern
Hemisphere (Howarth & Vandemark, 1989).
The intergenic region (IGR) of CLCrV
The integenic region (lGR) of CLCrV delimited by the flrst nucleotide of ACI and CP
ORFs is 352 nucleotide long. The IGR of all previously sequenced geminivirus DNAs
83
contain a sequence predicted to fonn a stable stem-loop structure. The equivalent stem-loop
structure, including the strictly conserved sequence TAAT A TT AC in its loop is also present
in CLCrV. This stem-loop structure is located between nucleotides 138 and 171. In addition
to the conserved stem-loop structure two inverted repeats and one direct repeat were
identified. Nucleotides at position 168 to 172 (CGCGC) is invertly repeated from 199 to 204
(GCGCG). Another inverted repeat is present at position 278 to 286 (CAACAACTT) and
294 to 302 (AAGTTGTTG). A direct repeat of 7 nucleotide (TGGAGCT) at position 57 to
63 and 64 to 70 is also present. Similarly positioned direct repeats have been implied as ACI
binding sites (Arguello-Astroga, et al., 1994). The intergenic region (lGR) of geminiviruses
plays very important role in the virus replication cycle. It includes the replication origin of
the geminiviral genome and two divergent promoters which differentially regulate the
temporal expression of viral genes (Haley et al., 1992; Lazarowitz et al., 1992; Sunter and
Bisaro, 1992). Cleavage site for geminivirus replication resides in the loop (Heyraud et al.,
1993 a,b). There are stretches of iterative sequence motifs of 8-12 nucleotide long, whose
organization is group specific (Arguello-Astroga, et al., 1994). In case of CLCrV this
sequence element appear to be 7 nucleotide long and 4 nucleotide upstream to potential
TA TA box which issimilar to the repeat elements in SLCV, BCMo V and PHV (Arguello
Astroga, et al., 1994)
Comparison of coat protein (CP) ORF
Coat protein (CP) ORF of CLCrV starts at nucleotide 353 and stops at nucleotide
1105 (Fig. 2), encoding a polypeptide of 251 amino acids with a molecular weight of 29.2
kd. The CP ORF, like those of several other WTGs, ends with two stop codons,
TAAT AA. The pair-wise comparison of CLCrV with other WTGs CP was done using
GAP program. Percent similarities and identities (in parenthesis) are shown in Table 2.
The CP is a highly conserved ORF among geminiviruses (Rochester et al., 1994) and is
84
essential for whitefly transmission (Stanley, 1985). CLCrV CP showed highest similarity
to that of Abutilon mosaic virus (AbMV) (96%) followed by PYMV and BDMV (95%),
SLCV and tomato mottle virus (ToMo V) (94%). The alignment of amino acid CP
sequences of different WTGs showed that there is a great degree of similarity among all
the viruses used in this study at several positions of the sequences (Fig. 3). There are
several blocks of 100% identical sequences. The potential motifs were searched for CPo
An A TP-GTP binding motif and zinc finger motif has been identified towards N-tenninal
of the ORF. Although both motifs contain one mismatch with the strictly defined motif
patterns they are highly conserved in all the geminiviruses used in the study (Fig. 3A).
There are a few substitutions at position 59-62 in zinc finger domain of some viruses. In
A TP-GTP binding domain valine is substituted by isoleucine in all of the 'Old World'
WTGs at position 112 except CLCuV, ICMV and one isolate ofTYLCV. On C-tenninus
of this ORF the sequences from amino acid 222 to 256 are 100% conserved, except at
position 232 where a leucine residue is replaced by a similar amino acid methionine in
ICMVand CLCuV (Fig. 3B).
The domains identified on this protein confirms the function of the protein previously
described (Azzam et al., 1994). The highly conserved regions are also in confirmation of
serological data, that WTGs share common epitopes (Harrison et al., 1991).
Comparison of replication associated protein (RAP, ACt) ORF
The AC1 ORF starts at position 2630 and stops at 1566 (Fig. 2). The total number of
amino acid residues are 355 and calculated molecular weight is 40.1 kd. The pair wise
comparison with counterparts in other gemini viruses is given in Table 2. This ORF shares
highest degree of similarity with SLCV (86%), followed by ToMoV (79%), PYMV (78%)
and BDMV (77%). All of these viruses are from the 'New World'. The percent similarity
to viruses like ICMV, CLV, TYLCV and CLCuV is 67, 69,70 and 72 respectively, which
85
is lower than that of 'New World' viruses. A perfect ATP-GTP binding domain was
identified towards the C-terminal portion at position 219 of the ORF. Alignment of this
portion (Fig. 3C) with other gemini viruses ORFs show that this domain is highly
conserved among all the WTGs used in this study. An imperfect topoisomerase II domain
is idntified at position 216, overlapping with ATP-GTP binding motif. There are a few
differences among the viruses at position 220 of this domain. The ACI is the only viral
encoded protein which is essential for replication (Elmer et al., 1988). The ACI protein is
related to replication initiator protein of eubacterial plasmids and is involved in rolling
circle mechanism of replication (Koonin and Dyina, 1992). In addition to an endonuclease
activity, viral DNA synthesis via rolling circle requires helicase and ligase activities.
Presence of ligase activity has not been addressed in geminiviruses, but a helicase activity
has been ascribed to the ACI protein. Its binding to elements in the IGR is 4 folds
stronger with ssDNA than dsDNA, a property common to many DNA helicases
(Thommes et al., 1993). The ATP-GTP binding site we and others (Gorbalenya et al.,
1990) identified may be a part of a putative helicase domain at the C terminus of ACI and
in ORF C2 of the CI-C2 protein.
Comparison of AC2, AC3, AC4 ORFs
AC2 ORF starts at nucleotide 1669 and stops at nucleotide 1250. The total amino
acid residues are 140 and the calculated Mr is 15.8 Kd. Amino acid sequence similarity is
given in Table 2. AC2 ORF of CLCrV showed highest similarity with BDMV (88%) and
PYMV (87%) and least similarity with CL V (61 %) and TYLCV -V (62%). The similarity
percentage with the other cotton geminivirus (CLCu V) was 69%.
AC3 ORF is located between nucleotide 1503 and 1105 and encode a protein of Mr
15.7 kd. Comparison of amino acid sequence of this ORF are give in Table 2. The ORF
C3 of CLCrV maintain highest sequence conservation after CPo CLCrV shares highest
sequence similarity in this ORF with AbMV (92%) followed by PYMV and ToMo V
(91 %) and BDMV (89%). The sequence similarity among 'Old World' viruses ranged
between 61 % (CLV), 62% (TYLCV-V), 69% (CLCuV) and 72% (TYLCV-M).
86
AC4 is a small ORF overlapping within the ORF ACl. It starts at nucleotide 2551 and
stops at nucleotide 2156. AC4 is 132 amino acids long with Mr of 14.8 kd. AC4 is the
least conserved ORF in tenn of sequence similarity amongst all the geminiviruses. The
amino acid sequence similarity of CLCrV ORF AC4 is gi-/en in Table 2. The biological
function of ORF AC4 is unclear. Mutations in the ORF AC4 of ACMV and TGMV had
no effect on infectivity and symptom development in Nicotiana benthamiana (Etessami et
a/., 1991; Elmer et a/., 1988), similar mutations in BCTV and tomato leaf curl (TLCV)
revealed the involvement of C4 in mediating the symptom severity in different host plants
and affected the virus movement in case ofTYLCV in tomato plants (Stanley & Latham,
1992; Rigden et a/., 1994; Jupin et a/., 1994). An mRNA transcript originating within the
ORF ACI was present in TLCV-infected tomato plants (Mullineaux et a/., 1993). These
studies suggest that ORF AC4 does exist but may playa different role in different virus
plant combinations.
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92
Table 1 Putative open reading frames (ORFs) in
cotton leaf crumple (CLCrV) DNA A
ORFs Reading Start Stop No. of Protein
Frame Amino Mr
acids
AVI +2 353 1105 251 29.2K
AV2 +3 2148 2469 108 11.8K
ACI -1 2630 1568 355 40.1K
AC2 -2 1669 1252 140 15.8K
AC3 -3 1503 1107 133 15.7K
AC4 -2 2551 2156 132 14.8K
AC5 -2 961 688 92 10.2K
A V and AC denote virion and complimentary sense for DNA A.ORFs respectively as shown in Fig.2
Table 2. Amino acid sequence similarities and identities, in parenthesis, (%) between
putative translation products of CLCrV With Other Geminiviruses
Virus Total (nt) CP ACI AC2 AC3 AC4
SLCV 73 94 (90) 86 (76) 74(62) 88 (77) 67 (54)
ToMoV 72 94 (88) 79 (67) 83 (75) 91 (83) . PYMV 73 95 (90) 78 (67) 87 (80) 91 (83) 35 (22)
BDMV 74 95 (91) 77 (67) 88 (83) 89 (80) 43 (25)
BGMV 70 92 (87) 74J63) 74(63) 87 (77) 36 (20)
TGMV 69 94 (89) 74 (61) 80 (67) 89 (80) 43 (24)
AbMV 73 96 (90) 73 (61) 80 (73) 92 (86) 39 (25)
BCTV . . 73 (55) - - 51 (30)
CLCuV 60 83 (73) 72 (55) 69 (52) 64 (49) 45 (25)
TYLCV(A) 60 84 (73) 71 (55) 64 (47) 64 (53) 40 (22)
TYLCV(V) 60 79 (71) 71(54) 62 (49) 69 (54) 36 (21)
CLV(N) 60 81 (71) 70 (55) 61 (46) 66 (54) 41 (27)
TYLCV(M) 60 78 (70) 70 (54) 72 (53) 64 (50) 40 (21)
PHV 65 93 (87) 70 (53) 67 (55) 78 (67) 37 (27)
CLV(K) 60 81 (72) 69 (55) 61 (45) 65 (53) 41 (26)
TYLCV(U) 59 80 (74) 69 (54) 71 (52) 65 (50) 40 (23)
ICMV 60 82 (73) 67 (52) 66 (52) 71 (52) 42 (23)
BCMV . . . . . 71 (61)
93
Note: Detennination of nucleotide (nt) identity and amino acid similarity among bipartite geminiviruses sequences was perfonned using GCG GAP program. A gap weight of 5.0 and a gap length weight of 0.3 were used for calculating nucleotide identity and a gap weight of 3.0 and gap length of 0.1 were used for amino acid comparisons Abbreviations for geminiviruses and their references: AbMV= Abutilon mosaic virus (Frischmuth et al .• 1990). ACMV= African cassava mosaic virus (Morris et al .• 1990), Bcrv=beet curly top virus (Stanley et al .• 1986), BDMV=bean dwarf mosaic virus (Hidayat et al .• 1992), BGMV= bean golden mosaic virus (Howarth et al .• 1985), CL V= cassava latent virus (Stanley et al .• 1983), ICMV=indian cassava mosaic virus (Hong et ai, 1993), PHV=pepper huasteco virus (Torres-Pacheco el al .• 1993), PYMV=potato yellow mosaic virus (Coutts el al .• 1991), SLCV = squash leaf curl virus (Lazarowitz el al .• 1991), TGMV= tomato golden mosaic virus (Hamilton el al .• 1984). TYLCV(A)= Tomato leaf curl virus (Dry el 01 .• 1993), ToMoV=tomato mottle virus (Abouzid el al .• 1992), TYLCV(V)= (Navot el al .• 1991), TYLCV(M) =tomato yellow leaf curl virus (Kheyr-Pour el al .• 1991) TYLCV(U)= tomato yellow leaf curl virus (Noris el al .• 1994)
94
.Figure 1. Complete nucleotide sequence of CLCrV DNA A. Nucleotide one of 2630 is
the fIrst nucleotide in intergenic region. Nine nucleotides (fAA TA TT AC) at position
152, which form the loop of a stem-loop region is conserved in all geminiviruses
examined. Sequence in bold are the intergenic region. Arrows indicate the stem in
conserved stem-loop structure.Italicized sequences are loop within the conservd stem
loop.
Figure 2. Proposed genomic organization for CLCrV DNA A. Arrows represent ORFs
in both orientations. IGR represents the intergenic region between ORFs ACI and A VI
which contain 34 base stem-loop structure found in all geminiviruses. ORF coordinates
are: A VI (CP) 253-1105; A V2 2148-2469; ACI 2630-1568; AC2 1669-1252; AC3 1503-
1107; AC4 2551-2156; AC5 961-686.
Figure 3. A). Alignment of capsid protein partial sequences showing consensus sequence
of an imperfect zinc fmger domain and an imperfect A TP-GTP binding motif (106-113).
Signature sequences for A TP-GTP motif and zinc fInger binding motif are shown at the
top of the figure. XX denotes any amino acid. Invariable amino acids for each motifs are
shown in bold. Consensus sequence for A TP-GTP domain is conserved in all the
geminiviruses listed. In zinc fmger domain the essential cysteins are conserved in all the
geminiviruses but histidine residue is not present in some of the viruses.
B) Alignment of amino acids from 218 to 256, showing consensus sequence in
capsid protein of all the geminiviruses used in the study. The sequence is 100% conserved
except alanine is replaced by glutamine at position 220 of two isolates of TYLCV and
leucin is replaced by methionine at position 132 in CLCuV and ICMV.
C) Alignment of amino acids from 222 to 232 of ACI protein, showing consensus
sequence for an A TP-GTP binding domain and a potential topoisomerase IT motif.
Signature sequences for ATP-GTP motif and topoisomerase IT (topoisomerase) motif are
95
shown on the top of the figure. 3. x denotes any amino acid. Invariable amino acids for
each motifs are shown in bold. Consensus sequence for this domain of ACt protein is
strictly conserved in all the geminivirus listed. The consensus sequence of geminivirus
ACt protein deviates from the topoisomerase II signature sequence by one amino acid
(alanine is replaced by arginine).
1 T~TTAAATA GTGACCCAGG ACTCAAAAAA TGCCTCTCAA CTTCTCTCAT 51 ATGTTGTGGA GTCTGGAGTC ~ATATAT GTTTACGCTA TAGAAGGCAT
101 TTGGGGACTC TCCTGGGCCC ACCATAGGAC TCCATACCQC GQCCATCTAT 151 ATAATATTAC TGATGQCCQC GCGGA'l"l"l'TG GAGCCCCC~ TTGTCGTTAG 201 CGCGACC~ AATTCAAATT AAAGGTTGCT TTTTTCTGTC GTCCAATCAT 251 TTTGCGCCTG ACTAGCCTAG ATATTAGCAA CAACTTCGCG ACTAAGTTGT 301 TGACCGTCAA GTATAAATTA ATGGGCTTAT GGCCCAATGG GTATAGAGCA 351 AAATGCCCAA GCGTGATGCC CCGTGGCGCT TATTAGCGGG TACGTCAAAG 401 GTAAGTCGAA ATGCCAACTA CTCTCCCTTG AGAGTATTGG GCCCTAAAGT 451 TAATAAGGCC TGCATATGGG TTAACAGGCC CATGTACAGG AAGCCCAGAA 501 TCTATCGCAC GTTAAGAACG CCTGACATTC CGAGAGGCTG TGAAGGGCCA 551 TGTAAAGTCC AGTCATATGA ACAGCGCCAC GATGTCTCAC ACGTGGGTAA 601 GGTAATGTGT ATATCTGATG TCACACGTGG TAATGGTATC TCCCTCCGTG 651 TCGGTAAGCG TTTCTGTGTT AAGTCTGTAT ATATTTTAGG GAAAATATGG 701 ATGGATGAGA ATATTAAGCT GAAGAACCAC ACGAATAGCG TCATTTTTTG 751 GCTGGTTAGA GATCGTAGAC CATATGGCAC TCCTATGGAC TTTGGTCAAG 801 TGTTTAATAT GTTTGACAAC GAGCCCAGTA CTGCCACTGT CAAGAACGAT 851 CTCAGAGATA GGTTGCAGGT CATGCACAAG TTTTATGCTA AGGTAACTGG 901 TGGACAATAC GCGAGCAACG AGCAAGCCTT GGTCAAGAGA TTCTGGAAGG 951 TCAACAATCA TGTGGTGTAC AACCACCAAG AAGCAGGGAA ATATGAGAAT
1001 CACACGGAGA ATGCGTTATT ATTGTACATG GCATGTACTC ATGCCTCTAA 1051 TCCTGTGTAT GCAACATTGA AAATTCGGAT CTATTTTTAT GATTCGACCA 1101 TGAATTAATA AAATTTAAAT TTTATTGAAT GATTTTCTAG TACATAACTG 1151 ACATACGACT TATCAGTTGC GAAATGAACA GCTCTAATTA CATTGTTAAG 1201 TGAAATTACG CCTAACTGGT CTAAGTACAA CATAACCAAA TGTCTAAACC 1251 TAATTAAATA AGTCGACCCA GAAGCTGTCA GAGATGTCGT CCAGACTTGG 1301 AAGTTCAGGT AAGCTTTGTG GAGATGCAAC GCTCTCCGTA AGTTGTGGTT 1351 GAATCGGGTC TGGACGTGGT ATATACGCGT CCTGGTGTAT AGCAGATCCT 1401 CTACTCCGTA TATCTTGAAA TAGAGGGGAT TTATTATCTC CCAGATATAC 1451 ACGCCATTCT CTGCCTGATG TGCAGTGATG AGTTCCCCTG TGCGTGAATC 1501 CATGACCGGT GCAGCCTATG TGGAAGTATA TGGTGCATCC ACAGTTTAGA 1551 TCAATGCGTC GTCGTCTAAT CGCCCTCCTC TTGGCTTGCC TGTGTGCCTT 1601 CTTGATAGAG GGGGGCGTCG AGAGTGATGA AGATAACATT CTTGAGTGTC 1651 CAATTTTTGA GCGACGCATT TTCCGCCTTA TCCAGGAAAT CTTTATAGCT 1701 GGATCCCTCG CCTGGATTGC AAAGCACGAT TGAAGGGATC CCACCTTTAA 1751 TTTGAACCGG TTTCCCGTAC TTGCAATTTG ACTGCCAGTC TTTTTGGGCC 1801 CCAAGAAGCT CTTTCCAATG CTTTAGCTTT AGGTATTGCG GTGCTACGTC 1851 ATCAATGACG TTATAGGCAA CATTATTCGA GTACACTCGA TTATTGAAGT 1901 CCAGGTGTCC ACTAAGGTAA TTATGTGGGC CTAAAGCACG AGCCCACATC 1951 GTCTTGCCTG TTCTAGAATC ACCTTCCACT ATGAGACTAA CTGGTCTTAC 2001 AGGCCGCGCA GGGGGACCCA TTCCAAAATA ATCATCTGCC CACTCTTGCA 2051 TCTGTTCAGG AACGTTAGTG AAAGAGGAAA GTGGAAATGG AGGGACCCAT 2101 TGTTCAGGCG CCTTTTTGAA AAGGCGTTCC AAGTTCGCTT GAATGTTATG 2151 GTAGCTCATG ATGAACGCCC TTGGATCTCC GGCCCGGATA ATTGCAAGAG 2201 CTTCCGCCAG ACTAGGTGCA TTGATGGCGT TATAATAGAC GTCGTCTTTA 2251 TTCTTCTTAG TACCCCTAGG CACTGTGAAC TGTCCGGATT CACAACAATC 2301 GCCGTCTTTG GTGATGTAAT TCTTGACGGC GTTGGCGTCT TTGGCTGCTT 2351 GTATGTTTGG GTGAAAATTG GTCGACCTTC TGGGGTGAGT AAGGTCGAAA 2401 AATCTCTCAT TCTTGATGTT GGACTTTCCA GAGAGTTGGA TGAGACAGTC 2451 AAGGTGTGGG AATCCGTCTG AGTGCTCCTC TCTGGCAACT CGAATAAATG 2501 TTGGTCTGAC GACTGACCAT GATAAGGTTT GAAGCATTTG AAGAGCTTCA 2551 TTTTTAGGGA TATCACACTT GGGATAGGTT AAGAAAATAT TTCTGGCTTG 2601 TAAACGAAAA GAATCGGCGT TTCTAGGCAT
Figure 1
96
Zinc Finger Atp-Gtp motif
Atp-Gtp GxxxxGKS
Zinc Finger cxxxxcxxxCxxxxxxxxHxxxH 63 106
Consensus D***GCEGPCKVQS*E*R*D*** GKRFC*KS PYMV ..... . DVPRGCEGPCKVQSFEQRHDILH .... GKRFCVKS BDMV ..... . DVPRGCEGPCKVQSYEQRHDISH .... GKRFCVKS ToMoV · ..... DVARGCEGPCKVQSFEQRHDISH .... GKRFCVKS ABMV · .... - DVPRGCEGPCKVQSYEQRHDISH .... GKRFCVKS CLCrV · ..... DIPRGCEGPCKVQSYEQRHDVSH .... GKRFCVKS SLCV · ..... DIPKGCEGPCKVQSYEQRHDISH .... GKRFCVKS TGMV · ..... DVPKGCEGPCKVQSYEQRHDISL .... GKRFCVKS BGMV · ..... DVPKGCEGPCKVQSYEQRHDISH .... GKRFCVKS PHV · ..... DVPKGCEGPCKVQSFEQRHDVSH .... GKRFCVKS ICMV · ..... DVPKGCEGPCKVQSFESRHDVVH .... GKRFCVKS CLCuV ..... . DVPKGCEGPCKVQSFESRHDVVH .... GKRFCVKS TYLCVA ..... . DVPRGCEGPCKVQSYEQRHDVAH .... GKRFCIKS CLVK ..... . DIPRGCEGPCKVQSFEQRDDVKH .... GKRFCIKS CLVN ..... . DIPRGCEGPCKVQSFEQRDDVKH .... GKRFCIKS TYLCVM ..... . DVPPGCEGPCKVQSYEQRDDVKH .... GKRFCIKS TYLCVU ..... . DVPFGCEGPCKVQSYEQRDDVKH .... GKRFCIKS TYLCVV ..... . DVPRGCEGPCKVQSYEQRDDIKH .... GKRFCVKS
Figure 3 (A)
218 256 PYMV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD BDMV .QEAGKYENHTENALLLYMACTHASNSVYATLKIRIYFYD ToMoV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD ABMV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD CLCrV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD SLCV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD TGMV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD BGMV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYVYD PHV .QEAGKYENHTENALLLYMACTHASNPVYATLKIRVYFYD ICMV .QEAGKYENHTENALMLYMACTHASNPVYATLKIRIYFYD CLCuV .QEAGKYENHTENALMLYMACTHASNPVYATLKIRIYFYD TYLCVA.QEAAKYENHTENALLLYMACTHASNPVYATLKIRIYFYD CLVK .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD CLVN .QEAGKYENHTENALLLYMACTHASNPVYATLKIRIYFYD TYLCVM.QEQAKYENHTENALLLYMACTHASNPVYATLKIRIYFYD TYLCVU.QEQAKYENHTENALLLYMACTHASNPVYATLKIRIYFYD TYLCVV.QEAAKYENHTENALLLYMACTHASNPVYATMKIRIYFYD
Figure 3 (8)
98
99
Atp-Gtp motif GxxxxGKT Topoisomerase VxEGDSAxG CONSENSES **EG*SR*GKT CLCrV lVEGDSRTGKT SLCV IIEGGSRTGKT CLV-N VIEGDSRTGKT CLV-K VIEGDSRTGKT TYLCV-A VIEGESRTGKT TYLCV-V VIEGDSRTGKT TYLCV-U VIEGDSRTGKT TYLCV-M VIEGDSRTGKT CLCuV VIEGDSRTGKT ICMV VIEGDSRTGKT PHV VVEGPSRTGKT BDMV lVEGDSRTGKT ToMoV lVEGDSRTGKT PYMV IIEGDSRRGKT TGMV IIEGDSRTGKT BGMV lVEGDSRTGKT AbMV lVEGDSRTGKT BCTV lVEGDSRTGKT
Figure 3 (C)
Chapter Four
Genomic Sequence and Phylogenetic analysis of Cotton Leaf Curl geminivirus DNA A
ABSTRACT
100
The complete nucleic acid sequence of a Pakistanian isolate of cotton leaf curl virus
(CLCuV) DNA A was detennined. The genomic DNA A consists of 2725 nucleotides.
Seven open reading frames (ORFs), two (A VI, and AV2) on the viral strand and five
(AC1, AC2, AC3, AC4, and AC5) on the complementary strand, capable of encoding
proteins with Mr greater than 10K can be predicted from the genomic sequence. With the
exception of AC5, the predicted genome organization is similar to those of whitefly-
transmitted, Old World geminiviruses. Computer-assisted sequence analysis and
comparison of the DNA sequence and predicted amino acid sequences for each ORF with
other geminiviruses showed that CLCuV maintained the greatest degree of similarity with
Indian cassava mosaic virus (lCMV), tomato yellow leaf curl virus (TYLCV) and cassava
latent virus (CLV), the viruses from 'Old World'. In cotrast, CLCuV showed lesser
similarity to squash leaf curl virus (SLCV), cotton leaf crumple virus (CLCrV) and
Abutilon mosaic virus (AbMV), all viruses from 'New World'. Phylogenies based on
distance and parsimony analysis confirm the divison of geminiviruses based upon
geographical proximity of the viruses.
Introduction
The geminiviruses are a group of plant viruses characterized by their distinctive
twinned isometric virion morphology (Harrison, 1985; Stanley, 1991), and circular ssDNA
genomes of about 2.5 to 2.8 kb. The International Committe on Taxonomy of Viruses
(lCTV) divided the geminiviruses into three distinct subgroups based on genome
101
organization, their insect vector and type of plant they infect (Hull, et 01., 1991).
Subgroup I contains the viruses that infect monocotylednous plants with maize streak:
virus (MSV) as the type member. These viruses are transmitted by leafhoppers and
contain a single DNA molecule. Subgroup II consist of viruses which are transmitted also
by leafhoppers and have single genomic DNA. The only members of this subgroup are
beet curly top virus (BCfV) and tobacco yellow dwarf virus (To YDV). Bean golden
moasic virus (BGMV) is the type member of the third subgroup comprising the whitefly
(Bemesia tabacl) transmitted geminiviruses with a bipartite genome and a host range of
dicotyledonous plants. Subgroup ill geminiviruses can be further divided into two subsets
according to gegraphical distribution (Howarth & Vandemark, 1989). The 'New World'
viruses, such as BGMV and tomato golden mosaic virus (TGMV) occurs naturally in
Western hemisphere. The 'Old World' geminiviruses such as African cassava mosaic virus
(ACMV) and tomato yellow leaf curl virus (TYLCV) occur naturally in eastern
hemisphere.
During 1991 and 1992, an outbreak: of a disease occured in cotton growing areas of
Pakistan. Leaves of infected plants curl upward. There is a marked swelling of leaf veins
with dark green color development. In addition, leaf like enations of various sizes,
originated in the leaf nectars, can be found on lower side of the leaf. Infected plants are
stunted and distorted, bearing few flowers. Yield may be reduced drastically depending
upon the time of infection. The disease is posing major production problems for cotton in
Pakistan (ICAC 1993). This disease has also been reported from Africa and Asia (Nour
and Nour, 1963). The symptoms on cotton are different than the one induced by cotton
leaf crumple virus (CLCrV), a geminivirus reported in North and Central Americas
(Brown et 01., 1987). In preliminary studies, geminivirus particles were found associated
with the diseased cotton plants (Mansoor et 01., 1993).
102
Recently, we reported the cloning of the DNA A component of CLCuV by a
polymerase chain reaction-based strategy (Chapter 2). The cloned viral DNA did not
hybridize with DNA of CLCrV under high stringent conditions. Partial sequence analysis
indicated that CLCu V is a whitefly-transmitted geminivirus distinctly different from
CLCrV. In this paper, we report the complete nucleotide sequence and the genome
organization of the CLCuV DNA A. The phylogenetic relationship based on different
ORFs of CLCuV with other geminiviruses is discussed.
Materials and Methods
Virus isolate and cloned DNAs
The CLCuV isolate used for cloning was originally collected in cotton growing area of
Multan, Pakistan. The virus was maintained and propagated on greenhouse cotton plants
at National Agricultural Research Center (NARC), Islamabad, Pakistan. The plasmids
containing the CLCuV DNA fragments, pCLCuVL and pCLCuVU were obtained as
described in Chapter Two. Unless stated otherwise, standard DNA manipulation
techniques as described by Sambrook et al. (1989) were followed. A set of nested,
overlapping subclones were generated by exonuclease III digestion (Henikoff, 1984) for
each clone. Subclones of approximately 150 ,.. 200 nucleotides apart were selected for
sequencing.
Nucleotide sequence determination
The complete nucleotide sequence of the DNA A was determined in both directions
using Sequenase II (United States Biochemical, Cleveland, OH) according to the
manufacturer's instructions and in the dideoxy-nucleotide chain termination reactions
(Sanger et al., 1977), using either 1'3 or T7 oligonucleotides as primers. Both DNA strands
were sequenced to ensure accuracy. Compression is some seqences were resolved by
replacing dGTP with dITP in sequencing reaction.
103
Sequence analysis
The total sequence of the viral DNA A was assembled with the GELASSEMBLE
program in the Wisconsin Genetic Computer Group (GCG) Software package. The
assembled sequences were initially compared with the available gemini virus sequences in
the Genbank and EMBL databases with FASTA program (pearson & Lippman, 1988)
available in the same software package. Determination of nucleotide identity and amino
acid similarity among bipartite gemini viruses sequences was perfonned using GCG GAP
program. A gap weight of 5.0 and a gap length weight of 0.3 were used for calculating
nucleotide identity and a gap weight of 3.0 and gap length of 0.1 were used for amino acid
comparisons.
Phylogenetic analysis
The sequences of untranslated intergenic region (UTR) and translated amino acid
sequences of different ORFs were aligned using PILEUP progarm in GCG package with
gap weight and length as describe above. Phylogenies based on unordered parsimony
were generated using version 3.1.1 of PAUP (Swofford, 1993), using default options with
a heuristic search. Inference of branch length was also conducted using parsimony
analysis in PAUP using ACCTRAN optimization. Regions at the begining and at the end
of multiple alignment was removed for phylogenentic analysis if majority of the aligned
sequeneces contained gaps. Divergence within the each group was considered on the
basis of average branch length.
Results and Discussion
Nucleotide sequence and ORFs ofCLCuV DNA A
The nucleotide sequences of clones pCLCuVL, pCLCuVU were determined from both
strands. Sequences derived from pCLCuVL and pCLCuVU corresponded to nucleotides
568 to1864 and nucleotides 1845 to 568, respectively. These two clones overlaped with
104
each other by 22 nucleotides at one end and 20 nucleotides at the other end. The
complete CLCuV DNA A component was determined to be 2725 nucleotides in length
and is shown in Fig. 1. The fIrst nucleotide in the intergenic region between the CP and
the ACI ORF was designated as nucleotide one of the circular DNA molecule. A total of
seven open reading frames in both the viral and the complementary strands with the
potential to encode proteins of Mr greater than 10 K were predicted (Table 1). The
genome organization derived from the sequence analysis is schematically represented in
Fig. 2. Two ORFs with products of 12.1 kd (A VI) and 29.6 kd (AV2) were predicted
from the virion strand of CLCuV DNA A. Both polypeptides are similar in amino acid
sequence and genome position to the precoat and capsid protein of other whitefly
vectored 'Old World' geminiviruses with dicotyledonous hosts. The complementary
strand is predicted to have 5 ORFs with potential to encode proteins of Mr greater than 10
K. The proteins were designated ACI (40.7K), AC2 (17.3K), AC3 (15.6K), AC4
(11.3K) and AC5 (19.4K) based on size, position and sequence similarities with
polypeptides of other gemini viruses (Tables 1 and 2). CLCu V DNA A encoded ORFs are
analogous to the ORFs VI, V2, Cl, C2, C3 and C4 in other geminiviruses (Dry et al.,
1993). The AC5 is an unique ORF and has not been reported for almost all other
geminiviruses. A similar ORF was reported in pepper huasteco virus (pacheco et al.,
1994), a whitefly-trasmitted geminivirus in Northern Mexico, and CLCrV, a cotton
geminivirus (Chapter 3). Re-examination of the nucleotide sequences of several other
viruses (BGMV, BDMV and PYMV) revealed a similar but smaller ORFs (pacheco et al.,
1994). Further genetic studies are needed to determine the role of this ORF. In
comparison with other whitefly transmitted geminiviruses (WTGs), the organization of
CLCu V DNA A is similar to, with the exception of one additional ORF in the
105
complementary strand, to the 'Old World' geminiviruses that infect dicotyledonous plants.
The genome organization of CLCu V is typical of WTGs from the Eastern hemisphere.
Comparison of entire sequence
Entire nucleotide sequence of CLCuV was compared with 16 whitefly geminiviruses
infecting dicots using GAP program of GCG package. The percent similarity is shown in
Table 2. The gemini viruses used in this comparison are grouped on the basis of their
geographic locations, the 'Old World', and 'New World' (Howarth and Vandemark,
1989; Abouzid et al., 1992). CLCuV showed greatest degree of similarity with Indian
cassava mosaic virus (ICMV) (74%) (Hong et ai, 1993), followed by tomato yellow leaf
curl (TYLCV) (73%) (Navot et al., 1991), cassava latent virus (CLV) (71%)(Stanley et
al., 1983), from the 'Old World', whereas it showed least homology with other
geminiviruses such as cotton leaf crumple (CLCrV) (60%) (Chapter 3), squash leaf curl
virus (SLCV) (60%) (Lazarowitz et al., 1991), bean dwarf mosaic virus (BDMV) (65%)
(Hidayat et al., 1992), and other geminiviruses from the 'New World'. This is a clear
indication that CLCuV belongs to the 'Old World' geminivirus group.
The intergenic region (IGR) of CLCuV
The intergenic region (IGR) of CLCuV is 421 nucleotide long. The IGR of all
previously sequenced geminivirus DNAs contain a sequence predicted to fonn a stable
stem-loop, also called structurally conserved element (SCE). The equivalent stem-loop
structure, including the sequence TAATATIAC in its loop is also found in CLCuV (Fig.
1). A stretch of 14 nucleotide (TGGCAATCGGTGTA) between nucleotide 3 to 16 was
directly repeated from nucleotides 30 to nucleotide 43. Another sequence element,
GGTGT, is repeated directly at nucleotide 11,38 and 61, and inversely (TGTGG)
repeated at nucelotide 173. The intergenic region (IGR) of geminiviruses is diverse
among all the geminiviruses (Lazarowitz, 1992), yet it plays very important roles in virus
106
replication and gene expression. It contains the replication origin of the geminiviral
genome and two divergent promoters which differentially regulate the temporal expression
of viral genes (Haley et 01., 1992; Lazarowitz et 01., 1992; Sunter and Bisaro, 1992).
Cleavage site for geminivirus replication also resides in the loop (Heyraud et 01., 1993a,b).
There are stretches of iterative sequence motifs of 8-12 nucleotide long, whose
organization is group specific (Astroga et ai., 1994). In case of CLCuV this sequence
element appear to be 14 nucleotide long and 11 nucleotide upstream to potential TAT A
box. The sequence element (AATCGGTGT) is directly repeated twice in TYLCV-I
(Astroga et 01., 1994). In CLCuV, this element is extended by four nucleotide at the 5'
end. A comparison of IGR sequences revealed that CLCuV is closely related to ICMV
and fonned a distinctive cluster with other bipartite WTGs from the eastern hemisphere.
This observation confirms that the grouping of geminiviruses, the 'New World' and 'Old
World' viruses, based on the geographical distribution (Howarth & Vandemark, 1989;
Abouzid et 01., 1992).
Comparison of A VI (precoat) sequences
The predicted products of the ORFs of different Old World and New World
geminiviruses that infect dicotyledonous plants were compared with the GAP program as
described in the materials and methods to determine percent similarity and identity
between the viruses as well as relatedness of the sequences within the group. The
percentage similarities and identities (in parenthesis) are shown in Table-2.
A small ORF, A VI, absent in the WTGs from the 'New World', is found in a similar
position in all Old World geminiviruses and overlaps the amino tenninus of the V2
(capsid) ORF. CLCuV maintain the highest percentage similarity in the AVI ORF with
ICMV (84%) followed by TYLCV-V (83%) and CLV (82%). The ORF VI is present in
all monopartite geminiviruses, and appear to be involved in virus spread in both
107
dicotyledonous (Khyer-Pour et al., 1991) and monocotylednous plants (Mullineaux et al.,
1988; Boulton et al., 1989).
Comparison of A V2 (capsid protein) sequences
The CP is a highly conserved ORF among geminiviruses with similarity varying
between 69 and 83% among the Old World geminiviruses (Rochester et al., 1994). The
New World geminiviruses maintain direct amino acid sequence similarities greater than
90% between SLCV, TGMV and BGMV sequences (Lazarowitz & Lazdins, 1991).
There are blocks of highly conserved amino acids throughout compared. C termini of the
Old and New World geminiviruses have very high degree of similarity. The sequences
from amino acid 220 to 246 (CLCu V ) is 100% conserved, except at position 230 instead
of leucine there is methionine in ICMV and CLCuV, both of which contain nonpolar
(hydrophobic) R groups (Fig. 3). The amino acid sequences of CLCuV CP showed
highest similarity to 'Old World' geminiviruses ranging from 96% in case of ICMV,
followed by TYLCV (89%), and CL V (88%).
Comparison of ACt ORF
ACI ORF starts at nucleotide 2725 and stops at nucleotide 1639 (Fig. 2). The total
number of amino acid residues is 361 and calculated molecular weight is 40.7 kd. A
comparison with other counterparts is given in Table 2. The ACI ORF shares highest
degree of similarity with different isolates ofTYCLV (89-88%) (Navot et al., 1991;
Khyer-Pour et al., 1991) followed by ICMV (87%) (Hong et ai, 1993),. All of these
viruses are from the 'Old World'. The percent similarity to viruses from the 'New World'
ranged from 72% in case of cotton leaf crumple virus (CLCrV) to 81 % in tomato mottle
virus (ToMoV) (Abouzid et al., 1992). The ACI ORF of PHV (Torres-Pacheco et al.,
1993), a virus from the 'New World', showed 84% sequence similarity with ACI ORF of
CLCuV. The ACI ORF of PH V is more conserved with the 'Old World' than 'New
World' viruses and maintain highest sequence conservation amongst four polypeptide
identified on complementary strand (Table 1). Rochester et 01. (1994) also reported
similar observation.
AC20RF
108
The percent similarity of AC2 ORF of the whitefly transmitted geminiviruses (WTGs)
to that of CLCuV is listed in Table 2. In summary, CLCuV AC2 ORF has the highest
percent similarity to the AC2 ORF of ICMV (77) followed by TYLCV -V, TYLCV -M and
CLV (75) and lower similarities with the viruses PHV (64%), AbMV, SLCV (66%) and
CLCrV (69%). The ORF AC2 encodes for a protein of about 17.3 kd. This gene in
bipartite geminiviruses encode a transcriptional activator that regulates the expression of
the CP and BVI genes (Haley et 01., 1992; Sunter & Bisaro, 1992).
AC30RF
The ORF AC3 shows the second highest conserved ORFs among the ORFs on the
complementary strand of the 'Old World' WTGs. The ORF AC3 of CLCuV shows
highest similarity to its analog in ICMV (81%), and least similar to AC3 ORF of PHV.
The comparison is listed in Table 2. The AC3 gene is considered to be non-essential,
however it increaes replication efficiency. AC3 mutants ofTGMV and ACMV
accumulated 50-fold less DNA (Etessami et 01., 1991; Sunter et 01., 1990)
AC40RF
The ORF AC4 is the smallest ORF in the CLCuV genome. This ORF has been found
in almost all the WTGs sequenced so far, but is often ignored because it does not fit the
criteria of potentially encoding for longer than 10 kd protein. The AC4 ORF in CLCuV is
100 amino acid long and can potentially encode for 11.3 kd protein. The AC4 ORF of
CLCu V is the least conserved among the geminiviruses we studied. It shares highest
109
similarity with BDMV (79%) followed by PHV (78%) and ICMV (76%) and the lowest
similarity to SLCV (40%) followed by CLCrV (45%) and CLV-K (56%)
Phylogenetic analysis
Phylogenetic analysis of each ORF and UTR resulted in one most parsimonious tree.
The trees are presented in Figures 3 A to G. Phylogenetic analysis of all ORFs separated
the whitefly-transmitted geminiviruses into two main groups, one correponding to the 'Old
World' group, and the other corresponding to 'New World' geminiviruses as defined by
Howarth & Vandemark (1989), except ORF AC4. The divergence within the two groups
were measured by using sum of total horizontal branch lengths within each cluster.
Divergence within the IGR and ACI ORFs ofthe 'New World' geminiviruses is more
than that of 'Old World' geminiviruses. CP ORF of the 'Old World' is more diverged
than the 'New World'. Other ORFs seem to be equally diverged. A phylogenetic tree
based on coat protein and replication associated protein sequences, shows that there is a
greater sequence divergence of monopartite geminiviruses as compared to bipartite
geminiviruses and 'Old World' viruses are more diverged than .the 'New World'
(fimmermans et al., 1994). Our analysis using all the ORFs show that the divergance
within each group varies with each ORF.
In the ORF AC4, the division of 'Old and New World' is violated. The reasons for
this exceptions may be; 1). This gene evolve so quickly that it does not leave any historic
records. 2). There is a horizontal transfer of gene. 3). The gene is evolving in a different
way that the present methods of analyses are not able to resolve the diversity.
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Table 1.
Putative open reading frames (ORFs) in CLCuV DNA A
ORFs Reading Start Stop No. of Protein Frame Amino
Mr acids
AVI +3 261 572 103 12.1K
AV2 +1 421 1191 256 29.6K
ACI -1 2725 1639 361 40.7K
AC2 -2 1744 1291 150 17.3K
AC3 -3 1599 1194 135 15.6K
AC4 -2 2575 2272 100 II.3K
AC5 -2 730 205 174 19.4K
A V and AC denote virion sense and complimentary sense
for DNA A ORFs respectively as shown in Fig.2
Table2.
Amino acid sequence similarities and identities (parenthesis) pecentage
between putative translation products of CLCu V With Other Geminiviruses
Virus Total nt) PRE CP ACI AC2 AC3 AC4
TYLCV(A) 72 76 (59) 89 (77) 89 (83) 72 (60) 78 (66) 74 (68)
TYLCv(M) 73 79 (65) 83 (74) 89 (81) 75 (56) 71 (57) 71 (64)
TYLCV(U) 72 - 86 (77) 88(79) 73 (55) 72 (59) 75_(68)
ICMV 74 84 (69) 96 (92) 87 (78) 77 (66) 81 (70) 76 (71)
TYLCV(Vt 73 83 (70) 86 (75) 85 (77) 75 (62) 80 (65) 64 (55)
CLV(N) 71 82 (64) 88 (76) 84 (78) 75 (62) 75 (64) 58 (50)
PHV 63 - 84 (74) 84 (72) 64 (49) 65 (48) 78 (69)
CLV(K) 71 82 (66) 88 (76) 83 (78) 75_(62) 75 (63) 56 (47)
ToMoV 65 - 82 (74) 81 (70) 70 (55) 66 (49) -BDMV 65 - 83 (72) 81 (69) 70 (56) 64 (51) 79(69)
TGMV 65 - 86 (75) 80 (70) 71 (56) 66 (52) 66 (56)
PYMV 64 - 83(73) 79 (69) 70 (55) 63 (47) 70 (58)
AbMV 63 - 84 (75) 79 (68) 66 (50) 65 (49) 70 (63)
BGMV 65 - 86(74) 78 (65) 70(55) 69 (52) 62 (54)
BCTV - - - 75 (63) - 62 (43) 66J58)
SLCV 60 - 84 (74) 72 (57) 66 (52) 63 (47) 40 (24)
CLCrV 60 - 83 (73) 72 (55) 69 (52) 64 (49) 45(25)
BCMV - - - - - - 5233)
115
.. Determination of nucleotide identity and amino acid similarity among bipartite gemimvlfUses
sequences was performed using GCG GAP program. A gap weight of 5.0 and a gap length weight of 0.3 were used for calculating nucleotide identity and a gap weight of 3.0 and gap length of 0.1 were used for amino acid comparisons.
Abbreviations/or geminiviruses and their re/erences: AbMV= Abutilon mosaic virus (Frischmuth el al., 1990). ACMV= African cassava mosaic virus (Morris el al., 1990). BCTV=beet curly top virus (Stanley el al .• 1986). BDMV=bean dwarf mosaic virus (Hidayat el al .• 1992). BGMV= bean golden mosaic virus (Howarth el al .• 1985). CLV= cassava latent virus (Stanley el al., 1983). ICMV=indian cassava mosaic virus (Hong el al. 1993). PHV=pepper huasteco virus (Torres-Pacheco el al., 1993). PYMV=potato yellow mosaic virus (Coutts el al .• 1991). SLCV = squash leaf curl virus (Lazarowitz el al .• 1991). TGMV= tomato golden mosaic virus (Hamilton el al .• 1984). TYLCV(A)= Tomato leaf curl virus (Dry el al .• 1993) • ToMoV=tomato mottle virus (Abouzid el al .• 1992). TYLCV(V)= (Navol el al .• 1991). TYLCV(M) =tomato yellow leaf curl virus (Kheyr-Pour el al .• 1991) TYLCV(U)= tomato yellow leaf curl virus (Noris el al .• 1994).
116
Figure 1. Complete nucleotide ofCLCuV DNA A sequence. Nucleotide one of 2725 is
the fIrst nucleotide in intergenic region. Nine nucleotides (T AAT A TT AC) at position
136, which form the loop of a stem-loop region is conserved in all geminiviruses
examined. Sequence in bold are the intergenic region. Arrrows indicate the stem in
conserved stem-loop structure.Italicized sequences are loop in the conservd stem-loop.
Figure 2. Schematic representation of CLCuV DNA A genome organization. Arrows
represent ORFs in both orientations. IGR represents the intregenic region between ORFs
ACI and AVI which contain 33 base stem-loop structure found in all geminiviruses. ORF
coordinates are: AVI (Pre coat) 261-572; AV2 (CP) 421-1191; ACI 2725-639; AC2
1744-291; AC3 1599-1194; AC4 2527-2272; AC5 730-205.
Figure 3. A-G. Dendrograms showing the relationships of whitefly transmitted
geminiviruses based on the nucleotide sequences of DNA A component and predicted
amino acid sequence A) Intergenic region from nt 1 to 250; B) Peptide sequence of ORF
CP; C) ORF ACl; D) AC2; E) AC 3;F) AC4; and G) A VI (pre coat). The sequences
were aligned by GCG program Pll..EUP and the relationship analysed by the PAUP
program (Swofford, 1993). The viruses are identifIed in the text and Table-I. Multiple
aligned sequences containing signifIcant gaps at the begining and at the end of alignment
were removed prior to phylogenetic analysis.
1 TTTGGCAATC GGTGTACACT CTAATTCTCT GGCAATCGGT GTAACGGGGT 51 GCAATATATA GGTGTACCCC AAATGGCATT ATCGTAATTT GAGAAATCAT
101 TTCAAAATCC TCACGCTCCA AAAAGCGGCC ATCCGTATAA TATTA~ 151 TGGCCGCGCG A'!"!"!'!"!"!"!"I'G TGGGCCCCCG ATTTATGAGA TTGCTCCCTC 201 AAAGCTAAAT AACGCTCCCG CACACTATAA GTACTTGCGC ACTAAGTTTC 251 AAATTCAAAC ATGTGGGATC CACTATTAAA CGAATTCCCT GATACGGTTC 301 ACGGGTTTCG GTGTATGCTT TCTGTGAAAT ATTTGCAACT TTTGTCGCAG 351 GATTATTCAC CGGATACGCT TGGGTACGAG TTAATACGGG ATTTAATTTG 401 TATTTTACGC TCCCGTAATT ATGTCGAAGC GAGCTGCCGA TATCGTCATT 451 TCTACGCCCG CGTCGAAAGT ACGCCGGCGT CTGAACTTCG GCAGCCCATA 501 CACCAGCCGT GCTGCTGCCC CCATTGTCCG CGTCACAAAA CAACAGGCAT 551 GGACAAACAG GCCCATGTAT AGGAAGCCCA GAATGTATCG GATGTACAGA 601 GGTCCGGATG TTCCAAAGGG TTGTGAAGGC CCATGTAAGG TACAATCTTT 651 TGAGTCGAGA CATGATGTCG TTCATATTGG TAAGGTAATG TGTATTTCTG 701 ATGTTACTCG TGGTGTCGGT TTGACCCATC GTATTGGTAA ACGTTTTTGT 751 GTCAAGTCAG TTTATGTTTT AGGTAAGATA TGGATGGATG AAAATATAAA 801 GACCAAGAAT CACACGAATT CGGTGATGTT CTTTTTAGTC CGCGATCGAC 851 GTCCTGTTGA CAAACCTCAG GATTTTGGTG AGGTATTTAA TATGTTTGAC 901 AATGAACCCA GTACAGCGAC TGTGAAGAAT AGTCATAGGG ACCr.TTACCA 951 GGTGTTGAGG AAATGGCATG CAACCGTTAC GGGTGGTCAA TATGCATCGA
1001 AGGAACAGGC TTTGGTCAAG AAGTTTGTCA GAGTTAACAA TTATGTTGTT 1051 TACAATCAAC AGGAAGCAGG AAAATACGAG AATCATACGG AAAATGCGTT 1101 AATGCTTTAT ATGGCTTGTA CTCACGCTAG CAACCCTGTT TATGCTACGT 1151 TGAAGATTAG GATATATTTT TATGACTCTG TAACGAATTG ATATTAATGA 1201 AGTTTGAATT TTATTTCTGA ATATTGATCT ACATACATAG TTTGTTGGAT 1251 TACATTGTAC AATACATGTT CTACAGCTTT AATAACTAAA TTAATTGAAA 1301 TTACACCGAG ATTGTTCAGA TATTTGAGGA CTTGGGTTTT GAATACCCTT 1351 AAGAAAAGAC CAGTCTGAGG CTGTAAGGTC GTCCAGATTC GGAAGGTTAG 1401 AAAACACTTG TGCAGTCCCA AAGCTTTCCG AGTGTTGTAG TTGAACTGGA 1451 TCCTGATCGT GAGTATGTCC ATATTCGTCG TGAATGGACG GTTGACGTGG 1501 CTGATGATCT TGAAATAAAG GGGATTTGGA ACCTCCCAGA TATATGCGCC 1551 ATTCCCTGCT TGAGCTGCAG TGATGGGTTC CCCTGTGCGT GAATCCATGG 1601 TTGTGGCAGT TGATTGACAG ATAATAAGAA CACCCGCATT CAAGATCTAC 1651 TCTCCTCCTC CTGTTGCGCC TCTTCGCTTC CCTGTGCTGT ACTTTGATTG 1701 GTACCTGAGT ACAGGGGTCT ATCAAGTGTG ATGAAGATCG CATTCTTTAC 1751 TGCCCAGTTC TTTAGTGCGG TGTTCTTTTC CTCGTCTAGG AATTCTTTAT 1801 AACTGCTGTT TGGACCAGGA TTGCAGAGGA AGATTGTTGG TATCCCWCCT 1851 TTAATTTGAA CTGGCTTCCC GTACTTTGTG TTGGATTGCC AGTCCCTTTG 1901 GGCCCCCATG AACTCTTTAA AGTGTTTGAG GAAATGCGGG TCGACGTCAT 1951 CAATGACATT ATACCAGGCG TCGTTACTGT AGACCTTGGG ACTCAGGTCC 2001 AGATGTCCAC ACAAATAGTT ATGTGGTCCC AATGACCTAG CCCACATCGT 2051 CTTCCCCGTA CGACTATCTC CTTCAATCAC TATACTTTGA GGTCTGTGTG 2101 GCCGCGCAGC GGCATCGACA ACGTTTTCGG AGACCCACTC TTCAAGTTCT 2151 TCTGGAACTT GATCGAAAGA AGAAGAAGAA AAAGGAGAAA TATAGGGAGC 2201 CGGTGGCTCC TGAAAGATTC TGTCTAGATT TGCATTTAAA TTATGAAATT 2251 GTAGTACAAA ATCTTTAGGA GCTAGTTCCT TAATGACTCT AAGAGCCTCT 2301 GACTTACTGC CTGCGTTAAG TGCTGCGGCG TAAGCGTCGT TGGCTGTCTG 2351 TTGTCCTCCT CTTGCTGATC TTCCATCGAT CTGAAACTCT CCCCACTCGA 2401 GAGTGTCCCC GTCCTTGTCG ATGTAGGCCT TGACATCTGA GCTTGATTTA 2451 GCTCCCTGAA TGTTCGGATG GAAATGTGCT GACCTGGTTG GGGATACCAG 2501 GTCGAAGAAT CTGTTATTCG TGCATTGGTA CTTCCCCTCG AACTGGATGA 2551 GCACGTGAAG ATGAGGTTCC CCATTTTGGT GAAGCTCTCT GCAGATCTTG 2601 ATGTATTTTT TGTTTACTGG TGTATGTAGG TTTTGTAATT GGGAGAGGGT 2651 TTCTTCTTTA GTTAGAGAGC ATTTGGGATA AGTGAGGAAG TAGTTTTTGG 2701 CGTTTGTTTT TAAAGCACGT GGCAT
Figure. 1
117
A
c
SLCV
r---L--- CLCrV
PYMV
iYLCV·Med
iYLVC.ls_(m)
TYLCV·lh
CLCuV
BDMV
PYMV
TGMV
~---BCTV
'----- PHV
CLCuV
iYLCVU
TYLCVM
TYLCW
CLCrV
'---- SLCV
Figure 3
B
o
ICMV
CLCuV
r------ TYLCV·A
CLV·K
CLV·N
,-----ICMV ,..----1
'----- TYLCV·A
'------ CLCuV
CLV·K
TYLCV·M
TYLCV·U
BGMV
'----- SLCV
L----TGMV
'-------- PHV
119
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