Molecular characterization of two cotton geminiviruses.

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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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read the dissertation prepared by Athar Nadeem -----------------------------------------entitled MOLECULAR CHARACTERIZATION OF TWO COTTON GEMINIVIRUSES

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Date

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.

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l11is dissertation has been submitted 'in partial fulfillment of requirements for an

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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

References

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Adejare, G. O. & Coutts, R H. A 1982. Ultrastructural studies on Nicotiana benthamiana tissue following infection with a virus transmitted from mosaic-diseased Nigerian cassava. Phytopalhol Z. 103,87-92.

Allen, R M., Tucker, H., & Nelson, R A 1960. Leaf crumple virus disease of cotton in Arizona. Plant Dis. Rep. 44,246-250.

Andersen, M. T., Richardson, K. A, Harbison. S. & Morris, B. A M. 1988. Nucleotide sequence of the geminivirus Chloris striate mosaic virus. Virology 164, 443-449.

Arguello-Astorga, G. R, Guevara-Gonzalez, R. G., Herrera-Estrella, L. R & Rivera­Bustamante, R. F. 1994. Geminivirus replication origins have a group-specific organization of iterative elements, A model for replication. Virology 203, 90-100

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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

50

CI JtepU.au.on

'" DNA aocumulaUoD.

DCI WO .... ~D.L;

drr-re::::.,:1.

~~-------~ CO

DK1 accYJUwaUoD

Figure 2

yz Coat prOLtlAl

"09II1II. •• 1.

V2 Coat , ... \dEl; Wcnoem-ot

,. 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.

• AC2

Figure 1

72

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

1·6

1·0

Figure 3

Figure 4

74

()

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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91

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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

ACl

G-(.~ D-t\->: ~ 2630 ~ CJ v Strand 71'

AC2

Figure 2

97

CP

AC5

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

ACl

G~~1)~ ~ 2725 ~ c.J V Strand ~

AC2 Figure 2

118

AVI (Pre coat)

AV2 (CP)

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

120

E F BGMV

SLCV SCLV

AbMV

ToMoV CLV·K

CLCrV

BOMV

TGMV BCTV

PYMV PYMV

PHV

TYLCV·U

TYLCV·M

TYLCV·U

TYLCV·V

TYLCV·A

ICMV TYLCV·A

CLCuV TYLCV·V

BCTV CLCuV

G CLVN

L..-- CLVK

TYLCV M

TYLCW

-ICMV

'---

-CLCuV

TYLC VA

Figure 3