MOLECULAR IDENTIFICATION AND COAT PROTEIN BASED GENOMIC CHARACTERISATION OF A VIRUS CAUSING YELLOW VEIN DISEASE ON CALENDULA

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BY AKILA. KHAN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2005

ABSTRACT

Calendula officinalis L. belongs to family Compositae/ Asteraceae which occupies the highest position among the angiosperms or at least among the dicots in the course of evolution. There are about 25 species of calendula but only two species viz. C. officinalis L. and C. arvense L. are found in India. Calendula is also known as pot marigold.

A new viral disease was observed on calendula at Aligarh Muslim University, Aligarh campus and in the surrounding area with the symptoms of yellowing of vein, s\\in\mg of plants, shortening of the \eaf and petioles, reduction in the nMmbei of flowers. Mosaic symptoms also appeared on calyx and corolla in severely infected plants. The incidence of disease was about 10-15% in nature. However, to provide clean and healthy environment of calendula to the pharmaceutical industries, it was essential to identify the causal pathogen and to develop the diagnostic at molecular level. So, virus free material may be made available.

The initial biological studies revealed that the causal pathogen of calendula was successfully transmitted from naturally infected calendula to the newly emerging seedlings of calendula through whitefly, which produces the symptoms alike to naturally infected plant.

The calendula isolate was tested for host range studies. The yellow vein disease of calendula was successfully transmitted to Nicotiana tabacum var. White Burley, Lycopersicon esculentum, and Capsicum annuum, producing leaf curling, shortening of intemodes and petioles. While Datura stramonium did not show any type of symptoms.

Based on symptomatology, host range studies and whitefly transmission, virus isolate causing yellow vein disease on calendula was suspected to be geminiviral in nature.

Virus isolate causing yellow vein disease on calendula, was further checked by PCR amplification with begomovirus specific TLCV CP primers. The PCR results gave positive amplification at ~750 bp in the plant showing symptoms of the disease. The specificity of the PCR amplicon was checked by Southern hybridization using radiolabelled probe of a well-characterized begomoviral (ITLCV), which proved that PCR amplification was geminiviral in nature.

The DNA band was LMP eluted and cloned in a suitable cloning vector (pGMT-T).

The sequence data of clone of calendula isolate revealed that the gene was 519 bp in length and it encompassed the complete ORf with initiation codon (ATG) to termination codon (TAA), which encodes 173 amino acid residues. The nucleotides sequence of the coat protein gene, was deposited in gene bank under the accession number (AY887174).

Amino acid alignment of calendula isolate revealed that four amino acid position "A2S", "K180R", "R180K" and "LKIILR-MRW218 to 227 YENHTENALM" were unique in the total amino acid sequence data. However, there were no unique sites from 61-120 and 241-253 amino acid.

Pairwise nucleotides alignment of the virus isolate from calendula with the selected geminivirus revealed maximum similarities 96% with Tobacco curly shoot virus (ToCSV) and Tomato Geminivirus-China (TomGV), minimiun similarities 77% with TLCV-Banglore. While pairwise amino acid alignment of the virus, isolate from calendula revealed maximum 97% similarities with TomOV-China. Based on pairwise alignment of nucleotides and amino acids i. e. maximum similarities 96-97% with ToCSV and TomOV-China. Virus isolated from calendula seems to be similar to that of TomGV-China and ToCSV geminivirus.

The dendrogram of nucleotides and amino acids showed close similarities of the virus isolate from calendula with ToCSV and TomGV-China.

The association of P-DNA molecule, which is one of the characteristics of several begomoviruses, was also checked by PCR using P-DNA specific universal primers. PCR product showed -650 bp amplification, half of the genome (1.3 kbp).

On the basis of results obtained by virus transmission, PCR amplification. Southern hybridization, sequence aligimient of nucleotides and amino acids and cluster analysis of CP gene, the virus has been identified as Calendula yellow vein virus (CYVV) which shows close similarity with Tobacco curly shoot virus and Tomato geminivirus-China. A P-DNA molecule has also been found to be associated with virus isolate from calendula. On the basis of above studies, the virus isolate from calendula comes under the subgroup of begomovirus, genus geminivirus and family geminiviridae.

Yellow net disease of calendula associated with Cucumber mosaic virus has been previously reported. However, our report is the first record on natural infection of calendula by a begomovirus and association of (3-DNA with virus isolate.

Moreover, the finding on molecular characterization of virus isolate causing yellow vein disease on calendula will open an understanding to the researchers on the genomic organization of a new begomovirus. Association of (3-DNA molecule with the virus isolate may have a definite role in symptoms severity or early development of symptoms as in case of Bhendi yellow mosaic disease. Tomato leaf curl disease, Cotton leaf curl disease.

The sequence data generated on CP gene of the virus may be utilized in developing CP mediated resistance against the virus isolate in calendula as well as other economically important plant. The clones generated during the studies may be used to develop molecular diagnostic probes for sensitive reliable detection of geminivirus on calendula, so that a healthy virus free plants may be made available to the pharmaceutical industries. MOLECULAR IDENTIFICATION AND COAT PROTEIN BASED GENOMIC CHARACTERISATION OF A VIRUS CAUSING YELLOW VEIN DISEASE ON CALENDULA

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BY AKIL A. KHAN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2005 '', ATSI* ;,>'>.

T6739 2402016(0) T^'Vil^l2721763(R ) 'amaz c/fb"0.i. ^^aqtiL 9412593463 MSc . Mphil, PhD, FPSI Plant Virology Lab, Deptt of Botany M N A Sc , CIES Pans Aiigarh Muslim University, Aligarh (U.P) INDIA

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This is to certify that the thesis entitled "Molecular identification and coat protein based genomic characterization of a virus causing yellow vein disease on calendula {Calendula officinalis L.)" submitted by Mr. Akil A. Khan to the Aligarh Muslim University, Aligarh for the award of the degree of Doctor of Philosophy, is a faithful record of the bonafide research work carried out in my guidance and supervision. He is allowed to submit this thesis for consideration of the award of the degree of Doctor of Philosophy in Botany (Plant Virology).

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Resi. : 332, Greater Azad Enclave Wesi, Dhorra, Angarh-202002 ACKNOWLEDGEMENTS

The author wishes to express his sincere gratitude to Professor Qamar Abbas Naqvi, Department of Botany, Aligarh Muslim University, Aligarh, for suggesting the problem and his constant advice, guidance and encouragement during the course of investigation and in the writing up of the manuscript. I am also greatly indebted to Prof. Ishrat Hussain Khan, Ex-Chairman, Department of Botany, Aligarh Muslim University, Aligarh for giving him permission to complete his work in National Botanical Research Institute, Lucknow. The author is also thanhful to Prof. Ainul Hague Khan, the present Chairman, Department of Botany, Aligarh Muslim University, Aligarh for his encouragement and help during the course of my research work. The author is deeply indebted to Dr. P. Pushpangadon, Director, National Botanical Research Institute, Lucknow to carry out different experiments at the Mol. Virology Lab of his Institute. The author is also so much thankful to Dr. S.K., Raj (Scientist F) for allowing him to perform various experiments in his Mol. Virology Lab and providing necessary facilities, without his help experimental work could not be materialized. I am highly thankful to all lab members and friends Sajid Khan, Rachana Singh, Sushil, Dharmandra, Atul, Arti, Radha, Sunil and lab assistants Jamal A. Ansari and Yashwant Singh for their constant help and encouragement during my experimental work in National Botanical Research Institute. I am also thankful to my lab colleagues Nuzhat, Tabasum, Shahidfor providing me their help and support. I am also extremely thankful to my friends Shiv, Irfan, Romana, Abid, Rafiq, Naeem, Faisal, Kashifand Manzarfor their constant support during my research work. The financial assistance given by A.M.U Aligarh in the form of University Fellowship is also gratefully acknowledge. The author is grateful to his parents and all the family members for providing him indispensable support and continuous encouragements during the research work.

(Akil A. Khan) DEDICATED TO MY BELOVED PARENTS CONTENTS

Page Nos.

1. INTRODUCTION 1-4

2. REVIEW OF LITERATURE 5-34

3. MATERIALS AND METHODS 35 - 59

4. RESULTS 60 - 67

5. DISCUSSION 68 - 71

6. SUMMARY 72 - 73

7. REFERENCES 74 - 97 ABBREVIATIONS

JO ACIDS

Ala (A) Alanine Arg(R) Arginine Asn(N) Asparagine Asp(N) Aspartic acids Cys (C) Cysteine Gin (Q) Glutamic acid His (H) Histidine He (I) Isoleucine Lue (L) Luecine Lys (K) Lysine Met (M) Methinone Phe(F) Phenylalanine Pro (P) Proline Ser(S) Serine Thr (T) Threonine Trp(W) Tryptophan Tyr(Y) Tyrosine Val (V) Valine

OTHER ABBREVIATIONS

Approximately Microgram Microliter ^M Micromolar AA Amino acid ATP Adenosine triphosphate Bisacrylamide N,N' methylene bisacrylamide pME Beta mercapto ethanol bp/kbp Base pair/ kilo base pair BSA Bovine serum albumine CCC Covalenty closed circular cDNA DNA complementary to RNA CLE Conserved late element CIAP Calf intestinal alkaline phosphatase CP Coat protein CR Common region CsCL Cesium chloride CTP Cytosine triphosphates DNA Deoxyribonucleic acid DNase Deoxyribonuclease dNTPs Deoxyribonucleotide ds Double stranded DTT Dithiothreitol EDTA Ethylene diamine tetra acetic acid EMBL European molecular biology laboratory Etbr Ethidium bromide G Gram GTE Glucose tris EDTA GTP Guanosine triphosphates Hr Hour IPTG Isopropyle-P-D-thioglactopyranoside IR Intergenic region L Litre LIR Large Intergenic region LA Luria agar LB Luria broth Mg Milligram Min Minute mM Millimolar M&M Materials and Methods MP Movement protein ng Nanogram NSP Nuclear shuttle protein nt Nucleotide NMR Nuclear magnetic resonance NW New World ORF Open reading frame OW Old Wold PCR Polymerase chain reaction PEG Poly ethylene glycol pmol Picomoles PVDF Polyvinylidenedifluoride RCR Rolling circle replication Rep Replicative protein REn Replication enhancer protein RNA Ribonucleic acid RDR Recombination dependent replication RT Room temperature RT-PCR Reverse Transcription Polymerase chain reaction SDS Sodium dedocyle sulphate S Second SE Sieve element Sat Satellite SIR Short Intergenic region ss Single stranded ssc Saline sodium citrate STE Saline Tris- EDTA TE Tris EDTA TAE Tri s-Acetate-EDTA TBE Tris borate EDTA TEMED N,N,N'N' tetramethyle ethylene diamine TrAP Transcription activator protein Tris Tris (hydroxymethyl) aminomethane TIP Thymidine triphosphates U Unit uv Ultra violet WTG Whitefly transmitted geminivirus X-gal 5-bromo-4-chloro-3-indolyle- P-D- glactopyranoside

VIRUSES NAME

ABMV Abutilon mosaic virus ACMV African Cassava mosaic virus AgBemoV Ageratum begemovirus AgEV Ageratum enation virus AYVV Ageratum yellow vein virus BCTV Beet curly top virus BeMYV Beet mild yellow virus BeWYV Beet western yellow virus BGMV Bean golden mosaic virus BYDV Bean yellow dwarf virus BYVV Bhendi yellow vein virus CLRV Cherry leaf roll virus CMV Cucumber mosaic virus CoLCV Cotton leaf curl virus CpeDV Chickpea chlorotic dwarf virus CYNV Calendula yellow net virus CYVV Calendula yellow vein virus DSV Digitaria streak virus EuLCV Euphorbia leaf curl virus ICaMV Indian cassava mosaic virus ITLCV Indian tomato leaf curl virus MSV Maize streak virus PaLCV Papaya leaf curl virus PHV Pepper huasteco virus PLCV Pepper leaf curl virus PLRV Potato leaf roll virus PVY Potato virus Y PYMV Potato yellow mosaic virus SqLCV Squash leaf curl virus TMV Tobacco mosaic virus ToCSV Tobacco curly shoot virus ToCSV-Y Tobacco curly shoot virus-Y ToCV Tomato Chlorosis virus ToLCV Tomato leaf curl virus-Banglore ToLCV Tomato leaf curl virus-Kamataka ToLCV Tomato leaf curl virus TomGV-China Tomato Geminivirus-China ToRV Tobacco rattle virus TPCTV Tomato pseudo curly top virus TuMV Turnip mosaic virus TYDV Tomato yellow dwarf virus TYLCV Tomato yellow leaf curl virus WDV Wheat dwarf virus WSBMV Wheat soil borne mosaic virus PREFACE

Plant viruses have had an impact on the science of virology ever since the discovery of Tobacco mosaic virus (TMV), at the end of the nineteenth century. Since than, the understanding of the genome organization of plant viruses has upgraded in parallel with the development of molecular biology and its related technique. A number of plant viruses have been characterized by biological, serological and molecular levels in the past one or two decades. The genus geminivirus belonging to the family geminiviridae, is one of the most economically important plant viruses that have been studied in the depth by several workers from all over the world. Geminivirus is an paired, twin moon shape having geminate particle about -3.0 kbp in diameter and whitefly transmitted virus. It has the widest host range. This dissertation is devoted to the biological as well as molecular characterization of an virus isolated from Calendula officinalis L. and its compression with other reported geminiviruses, especially with reference to the coat protein gene. In this research study, the objectives of the work undertaken, technique used, result obtained and conclusion drown are incorporated under the following headings. 1. INTRODUCTION:- Chapter one commences with a brief introduction to the background and objective of the problems followed by a relevant review of literature. 2. REVIEW OF LITERATURE:- This chapter reviews the various studies carried out till date in order to understanding the genomic characterization of geminivirus and reported virus on calendula. 3. MATERIALS AND METHODES:- This chapter describes the experimental procedure and technique that were employed in order to accomplish the objective of the dissertation. 4. RESULTS:- Details of various observation made and result obtained from the experiments that were performed are described in this chapter. 5. SUMMERY:- This chapter summarizes the work that has been presented in this dissertation and conclusion drawn from it. 6. BIBLIOGRAPHY:- This chapter lists the publication that have been referred to in the dissertation. Introduction INTRODUCTION

Plant viruses are intracellular parasites that have a small genome made up of RNA or DNA. At present, viruses are classified into 184 genera and of these 161 are classified in 54 families. Only 11 families and 34 genera infect plants. The losses caused by plant viruses in the tropical and subtropical regions are of greater significance as they provide ideal conditions for them and their vectors to perpetuate. The tropical location of Indian subcontinent with its diverse climatic conditions ranging from high rainfall areas to deserts on one hand and snow - capped mountains to peninsula on the other, provide a large number of viral diseases to flourish thus affecting various cropping systems. However, in nature, once infected, viruses can not be controlled by any chemical means, therefore, disease management must be based on strategies that prevent infection of plants in the field as there is no cure of infected plants.

Virus infections cause huge losses to all economically important plants. Of all known plant viruses around 70% are transmitted from plant to plant by invertebrate mobile vectors such as aphids, leafhoppers, whiteflies, mites, thrips and beetles etc. Insect mediated transmission is the most common mode of transmission of plant viruses in nature. Aphids alone transmit about 290 plant viruses among all insect vectors.

Plant pathogenic viruses mostly have ribonucleic acid (RNA) as their genetic material but a few viruses have de-oxyribonucleic acid (DNA) as their genome. Geminiviruses, belonging to family Geminiviridae, are a group of DNA viruses that are classified into four genera- mastrevirus, curtovirus, begomovirus, and topocuvirus based on genomic organization, insect vector and host range. They are characterized by having geminate morphology of particle and single stranded genome comprising of one or two DNA components that are <3kbp in length.

The diseases caused by geminiviruses are of utmost concern in the tropical and sub-tropical areas of the world. A few of them have hampered the economical cultivation of several legumes (beans, soybeans, pigeonpea and other edible legumes), vegetables (potato, brinjal, tomato, okra, chillies), sugar crops (beet), cereals (maize and wheat). In the past 15-20 years, both prevalence and distribution of whitefly Introduction

transmitted geminiviruses (WTGs) have increased and the impact has been devastating. Depending on the crop, season, whitefly prevalence and other factors, yield losses range from 20-100%. The WTGs have thus become a major group of pathogen of vegetables in the tropical and sub-tropical regions.

More than 80% of the known geminiviruses are transmitted by whiteflies and belong to the genus Begomovirus, which mostly have bipartite genomes designated as DNA-A and DNA-B and infect dicotyledenous plants although numerous begomovirus with a monopartite genome occur in the Old World,

Economic losses due to geminiviruses infection in cassava are estimated to be US$ 1300-2300 billion in Africa (Thresh et al., 1998), US$5 billion for cotton in Pakistan between 1992-97 (Briddon and Markham, 2000), US$300 million for grain legumes in India (Verma et al., 1992) and US$140 million in Florida for tomato alone (Moffat, 1999). Despite concerted efforts to contain geminiviruses and their vectors, menacing disease epidemics caused by newly emerging or re-emerging geminiviruses are becoming frequent and appearing even in new regions, previously free from such diseases. The frequency with which new begomoviruses are appearing shows that these viruses are still evolving and pose a serious threat to sustainable agriculture, particularly in the tropics and sub-tropics. In recent years, some begomoviruses have also moved to temperate regions causing concern in the production of vegetables in greenhouses. Another concern is the emergence of diseases caused by a complex of begomovirus satellite DNA molecules.

Geminiviruses, member of family Geminiviridae, which cause several plant diseases of great importance, have been increasing rapidly since somewhat belated discovery of their characteristic geminate (twined) particles in 1974 (Bock et al., 1974) and of their circular single stranded genomic DNA in 1977 (Goodman et al., 1977; Harrison et al., 1977). Indeed they have become one of the best studied families of plant viruses and have been shown to possess several unique features.

Rosette disease of calendula was transmitted by whiteflies in nature and could be transmitted by grafting but not by sap extracted from infected leaves or by aphids (Gupta and Verma et al., 1983). The calendula plant has also been found susceptible to infection of Tomato chlorosis virus (ToCV) of the genus Crinivirus in the family Introduction 3

Closteroviridae, which has broad host range as it includes 24 plant species in 7 plant families. (Gail et al,. 1999). It is prevalent everywhere, particularly in tropical and sub­ tropical areas of the world. Climate in these areas is conducive to ornamental crops as well as favourable for the growth and spread of the vector whitefly. Initially the disease was restricted in the tropical areas of the world but due to import of the seedlings (which might be accompanied by the virus and its vector) to many countries in.the temperate regions, the disease has spread to these areas too with the whitefly becoming adapted to the green house conditions.

The climate in India is also favourable for growth of the host and the vector. The disease was first reported on Calendula officinalis L. as mixed infection of two viruses i.e. Cucumber mosaic virus and Turnip mosaic virus by Lisa et al. in 1979. Purification and properties of Calendula yellow net virus was reported by Naqvi et al., 1985 who placed Calendula yellow net virus under cucumovirus group. A strain of Tobacco mosaic virus from Calendula officinalis L. was isolated by Hristova et al. 1994. They found indistinct mosaic symptoms on green house grown C. officinalis L. Based on host range studies, serological and electron microscopic examination, the virus isolated from C. officinalis L. was identified as Tobacco mosaic Tobamovirus.

Calendula offiicinalis L. belongs to family Compositae / Asteraceae, which occupies the highest position among the angiosperms or at least among the dicots in the course of evolution as is evident by the presence of largest number viz., 2000 species of 950 genera. There are about 25 species of calendula but only two species i.e. C officinalis and C. arvense L. are found in India. Genus calendula is distributed in South Europe, Northern Africa and Western Asia and is grown in temperate regions around the world. Calendula is easily propagated by seeds. The plant is annual or perennial herb with 1-2 feet height. The ligulate floret are bright, orange or yellow in color. (Wealth of India).

Economic importance of calendula

Calendula has great economic importance. The flowers contain an amorphous bitter principle calendulin, a yellow tasteless substance analogous to bassorin, trace of an essential oil, alenolicacid, a gum, a sterol (C26H44O2 m.p. 209°) cholesterol, esters of lauric, myristic, palmitic, stearic and pentadecyclic acid faradiol m.p. 211° and amidiol Introduction

m.p. 257°. The pigment is a mixture of P-carotene, lycopene, viola-xanthin and other xanthophyll. Calendula officinalis L. also known as pot marigold, is a mild aromatic with diaphoretic, diuretic and stimulant properties. A mixture of dried flowers is administered in the treatment of amenrrhoea and after dilution as a lotion for sprains and bruises. Calendula is now almost absolute as a drug and is used to adulterate saffron and arnica flowers. (Wealth of India).

In India, calendula extract has been used for antiseptic cream named as (borocalendula) by many private pharmaceutical industries. It is used for the treatment of cracks, sunburn, lips protection and in the cure of skin diseases.

Like other pathogens that affect the aesthetic value of ornamental plants, viruses are of significant importance due to absence of therapeutic control measure against them in plants. Viruses have considerable economic importance in floriculture due to severe losses, reduction in growth, number and size of flowers caused by them. Thus, viruses are a potent threat as well as limiting factor in floriculture industry.

A new viral disease was observed on calendula at Aligarh Muslim University, Aligarh campus and in the surrounding areas with the symptoms of yellowing of vein, stunted growth of plants, shortening of the leaf and petioles and reduction in the number of flowers. Mosaic symptoms also appeared on calyx and corolla in severely infected plants. The incidence of disease was low about 10-15% in nature. However, to provide clean and healthy plants of calendula to the pharmaceutical industries, it was essential to identify the causal pathogen and to develop the diagnostics at molecular level. So that virus free material may be made available.

Aims and Objectives

1> Biological characterizations of virus isolate. 2> Molecular characterization of virus isolate. 3> Detection of virus by molecular diagnostic tools. Review of Literature GEMINIVIRUSES

Geminiviruses are major plant pathogens in tropical and subtropical countries. Due to very low titers, tight phloem association, poor or no mechanical transmission, fragility and lack of local lesion host, the geminivirus remained obscure and uncharacterized for a long time. Geminiviruses are a diverse group of plant viruses and they derive their name from the characteristic paired / geminate virion particle (Bock et ah, 1974 and Zhang et al., 2001), and have a characteristic double icosahedral ("twin moon", hence Gemini) capsid of approximate dimensions 18x30 nm (Esau, 1977). The capsid contain a covalently closed circular single stranded DNA 2.5-3.0 kb in size (Goodman, 1977, Goodman et al, 1977, Harrison et al., 1977, Hatta and Francki, 1979, Reisman et al, 1979, Francki et al, 1980). All geminiviruses are now included in Geminiviridae family (Mayo, 1996).

The Geminiviridae family includes a large number of plant viruses that produce significant losses in economical important crops of both monocotyledonous and dicotyledonous plants (Moffat AS, 1999).

Transmission of the disease

Persistent nature of transmission of geminiviruses by the whiteflies was shown by (Butter «fe Rataul 1977).

Virus - vector relationship

The virus - vector relationship has been extensively studied by several workers (Butter & Rataul 1977, Reddy and Yaraguntaiah, 1981) and information on some of the aspects of this relationship is given as:

Acquisition and inoculation threshold

When non-viruliferous whiteflies were given acquisition feeding for the specified periods followed by acquisition access period of 12 hr or 24 hr it was observed that a single female whitefly could become viruliferous after 31 min of feeding on a test plant resulted 8% transmission and a prolonged acquisition access of 24 hr resulted in 30% transmission. The whiteflies that were allowed access on a Tomato Leaf Curl Virus source were used to determine the inoculation threshold period. A minimum inoculation-feeding period of 32 min was required by a single Review of Literature 6

whitefly to cause infection on tomato test plants and gave 4% transmission. An inoculation access of 24 hr increased the transmission of Tomato Leaf Curl Virus by 42% with a single female whitefly per plant (Butter and Rataul, 1977, Reddy and Yaraguntaiah, 1981).

Effects of pre-acquisition as well as pre-inoculation fasting

Tomato Leaf Curl Virus transmission of upto 36% was obtained when the whiteflies were starved for one hr prior to acquisition. On increasing the fasting time lower transmission rates were obtained. Efficiency varied with sex of the whitefly, female being more efficient than males (Varma, 1952, Cohen and Nitzany, 1966, Rathi and Nene, 1974). Like wise higher transmission of up to 40% was obtained when viruliferous whiteflies were starved for one hr prior to inoculation as compared with unfasted whiteflies. The transmission rates dropped sharply when the pre-inoculation fasting period was increased beyond one hr. Acquisition and inoculation efficiency was also affected by the leaf on which the whiteflies fed. Highest efficiency was obtained when the whitefly fed on the youngest leaf of the plant. Further whitefly colonies in which eggplant was the rearing host, were found the most efficient as compared to other rearing hosts such as tobacco and cotton (Reddy and Yaraguntaiah, 1981).

Taxonomic Position of geminivirus

A taxonomic structure for the Geminiviridae family, based on sequence comparison and biological properties of geminiviruses was proposed by Padidam et al, 1995b. According to this scheme, family Geminiviridae has been divided into alphageminivirinae and betageminivirinae subfamilies (Fig.2.1). Subfamily alphageminivirinae consists of genus monogeminivirus consisting of leafliopper transmitted geminiviruses infecting monocots or dicots and having a single genomic component. Type member is Maize streak virus (MSV) and if one compares this with older classification scheme, members of subgroup I fall in this genus. On the other hand, subfamily betageminivirinae is classified into three genera namely Intergeminivirus, Arceogeminivirus and Neogeminivirus. Genera intergeminivirus includes viruses, which are leafliopper transmitted, infect dicots and have a single genomic component. Archeo and Neogeminivirus genera include whitefly transmitted geminiviruses (WTGs), the former encompasses WTGs from the Old World (OW), can CQ O 3 cfl « O Ip c to o O e -o 2 o o ^- ^ > > > o > Q a- O X3 Z 1 82 i in .— c t- O O ON J= o o = > > >

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have either one or two component genomes and infect dicots whereas the latter is occupied by those WTGs which belong to the New World (NW) (Table 2.2). All the WTGs characterized from the NW have been found to have two genomic components.

The geminivirus group was upgraded to the family Geminiviridae with three genera namely Mastrevirus, Curtovirus and Begomovirus in 1995 (Murphy et al., 1995). It includes a large number of plant viruses that produce significant losses in economically important crops of both monocotyledonous and dicotyledonous plants (Moffat AS, 1999). Fourth genus in Geminiviridae was approved by International Committee on Taxonomy of Viruses (ICTV) in 1999 (Faquet and Stanley, 2003). Therefore, at present, the family Geminiviridae has four genera viz. Mastrevirus, Curtovirus, Topocuvirus and Begomovirus, which differ mainly with respect to transmission of vector, genomic components and host range (Harrison et al, 2002, Gutierrez, 2002). The name of the genera are derived from the names of their type species. The genus Mastrevirus is transmitted by leafhoppers, generally infects monocots and has monopartite genome (such as type species: Maize streak virus, MSV). Curtovirus is leafhopper - transmitted, infects dicots and has either monopartite or bipartite genome (Beet curly top virus, BCTV). The genus Topocuvirus is transmitted by treehoppers, infects dicots and has monopartite genome {Tomato pseudo curly top virus, TPCTV). Finally, Begomoviruses are whitefly transmitted, infect dicots and have bipartite genome (Type species: Bean golden mosaic virus - Puerto Rico, BGMV - PR, African cassava mosaic virus, ACMV, Squash leaf curl virus, SqLCV). However, some begomoviruses like Tomato yellow leaf curl virus, TYLCV have monopartite genome that resembles DNA-A (Gutierrez, 2002:; Verma and Malathi, 2003) (Table 2.1).

Geminiviruses cause significant yield losses to many crop plants throughout the world. Because of their economic importance and the relative ease with which their DNA genomes can be cloned. Many geminiviruses have been isolated and characterized. The problem is compounded by the recent discovery of a high frequency of recombination between species of geminiviruses. Guidelines for naming geminiviruses have been proposed and accepted by the Study Group and the geminivirologist community as a whole. (Fauqet et al, 2003). These provide a means of distinguishing viruses that are sufficiently distinct to be classified as distinct species. CO a, E o > o > _>% >. U o E CO 3 o S CO CJ c CO O o C/) O X H 6 u •*-es> > c 3 u c a CO (—1 o s o Cu s ••—» o> e CO 3 o u C/5 o ti a (U >. N § O 1 3 E "3 m H > E H o o ^•B ns c t/5 o •*-o» 4-) o CO CO CO ra o •*-» c c O O •*—O' k. O o o 0) O o 4^ c a ^^ >» o n •a • 0) o> •4-» •*-» 4-1 (0 CO t: o o 1 CO CO Q. o H a, •ti D, &, o cs o O o C o o ^ G c c CO C c Q. >» o o cu o o 0) o c s £ a n r :s 6 >s S

tn k4 i-i o6 aj E ex Cu n. 4-i ex o. Cu 5 (1) o o o 0) 3 o Xi re u re CO CO i_ UL > u u H > •J >« c ^ E "O 0) 0) O o 0) a ^—H >^ £ o 1- u ^^-^ CO Q. CO CO S 3 CM M a 5 s > •> 0) o 0) (0 s 3 h. u > o CJ 3 6C ^ k •*-» o S o re CO o o, 1- > 3 CO o V3 o OQ H Table2.2 Members of the subgroup III of Geminiviridae family (various characterized geminiviruses).

VIRUS NAME LOCATION REFERENCE

African cassava mosaic virus, ACMV Old World Stanley and Gay, 1983 Tomato golden mosaic virus, TGMV New World Hamilton et al., 1984 Abutilon mosaic virus, ABMV New World Frischmuthetal.,1990 Potato yellow mosaic virus, PYMV New World Couttsetal.,1991 Squash leaf curl virus New World Lazarowitz and Lazdin,1991 Tomato yellow leaf curl virus,Israeli Old World Navotetal., 1991 isolate Tomato yellow leaf curl virus, Old World Kheyr-Pouretal,.1991 Sardinian isolate,TYLCVS Tomato mottle virus, TMoV New World Abouzid et al,. 1992 Bean dwarf mosaic virus, BDMV New World Hidayat et al., 1993 Bean golden mosaic virus Brazilian New World Gilbertsonetal,. 1993 isolate, BGMVB Pepper huasteco virus, PHV New World Torres-Pacheco et al,.1993 Indian cassava mosaic virus, ICMV Old World Hongetal,. 1993 Mungbean yellow mosaic virus, Old World Morinaga et al,. 1993 MYMV Tomato leaf curl virus, Australian Old World Dryetal,. 1993 isolate, TLCVA Tomato leaf curl virus, Spanish isolate Old World Moriones et al,. 1993 (from Almeria), TYLCVA Tomato leaf curl virus. South Indian Old World Chatchawankanphanich et al,. isolate, TLCVSl and TLCVS2 Hong and Harrison, 1995 (TLCV2) Bean golden mosaic virus, Guatemalan New World Fariaetal,. 1994 isolate, BGMVG Tomato yellow leaf curl virus, Thailand Old World Rochester et al,. 1994 isolate, TYLCVT Tomato yellow leaf curl virus, Sicilian Old World Crespietal,.1995 isolate, TYLCVL Ageratum yellow vein virus, AYVV Old World Tenetal,. 1995 Tomato leaf curl virus, Indian isolate, Old World Padidam et al,. 1995a ToLCV-India Review of Literature 8

However, guidelines for, as outlined in the Seventh Report of ICTV, are no longer precise enough to demarcate some species and consequently require urgent revision.

The criteria for determining the taxonomic status of virus in the Seventh Report of the ICTV demarcating geminiviruses species is summarized as follows:

> Different number of genome components.

> Different organization of genes in the genome.

> No trans-complementation of genes products.

> No pseudorecombination between components.

> Nucleotide sequence identity (<75% for mastreviruses, <80% for curtoviruses and <90% for begomoviruses).

> Virions react differently with key antibodies.

^ 90% coat protein gene sequence identity.

> Different vector species.

> Different host range pathogenicity.

Because of the large number of begomoviruses that have been isolated, these criteria are becoming less reliable for distinguishing species and strains. The molecular criteria probably have the most practical value.

Variability in geminiviruses has arisen through mutations, recombination and pseudorecombination. Genomic recombination in geminiviruses, not only between the variants of the same virus but also between species and even between genera, has resulted in rapid diversification. Heterologous recombinants containing parts of the host genome and or sequence from satellite-Jike molecules associated with monopartite begomoviruses provide unlimited evolutionary opportunities. Human activity has also played an important role in the emergence of serious geminivirus diseases across the globe, like the changes in cropping system, the introduction of new crops, the movement of infected planting materials and the introduction of host susceptibility genes through the exchange of germplasm.

Geminiviruses constitute the second largest family of plant viruses, the Geminiviridae, comprising of four genera, Mastrevirus, Curtovirus, Begemovirus and Review of Literature

Topocuvirus. During the last two decades, these viruses have emerged as devastating pathogens, particularly in the tropics and subtropics, causing huge economic losses and pose threat to crop production. Epidemics caused by re-emerging and newly emerging geminivirus are becoming frequent even in regions that were earlier free from theses viruses. As compared to mastreviruses and curtoviruses, begomoviruses have emerged as more serious problems in a variety of crops, for example, cassava, cotton, grain, legumes and vegetables. Major contributory factor for the emergence and spread of new geminivirus disease are the evolution of variants of the viruses, the appearance of the whitefly 'B' biotype and the increase in the vector population variability in geminivirus has arisen through mutations, recombination and pseudorecombination. Genomic recombination in geminivirus, not only between the variants of the same virus but also between species and even between genera, has resulted in rapid diversification. From the disease point of view, most virulent variants have developed through recombination of viral genomes such as those associated with cassava mosaic, cotton leaf curl, and tomato leaf curl diseases.

Geminiviruses have either one or two ssDNA molecules ranging from 2500 to 3000 nucleotides in size as their genome. The open reading frames (ORFs) are specified in either virion sense strand (V) (the strand that gets encapsidated in virion particle) or complementary sense strand (C) and are transcript bi-directional.

The geminivirus genome consists of single stranded DNA (ssDNA), either one or two small circular molecule, although double stranded DNA (dsDNA) forms appear as replication intermediates. The geminivirus replication cycle relies entirely on DNA intermediates and occurs within the nucleus of the infected cell through two basic stages:

(i) Conversion of ssDNA to dsDNA intermediates,

(ii) Rolling circle replication (RCR) (Crisanto Gutierrez 2002).

Satellites are a common feature of a number of RNA viruses. These molecules are classically defined as viruses or nucleic acids that depend on a helper virus for replication, are dispensable for replication of helper virus, and lack sequence homology to the helper virus genome (Murant and Mayo, 1982.) Diversity of ^-DNA, a satellite molecule associated with some monopartite begomoviruses (Briddon et al, 2003). Review of Literature 10

A single begomovirus, Tomato leaf curl virus (ToLCV), originating from Australia, was shown to be associated with a single stranded DNA satellite molecule. This satellite had no discernible effects on either viral replication or on the symptoms caused by ToLCV (Dry et al, 1997). The ToLCV satellite is approximately 682 nt. in length and sequence unrelated to ToLCV and requires ToLCV for replication, spread in plant and insect transmission. Recently satellite molecules related to the ToLCV-sat were identified for several other monopartite begomoviruses. These molecules, called as p-DNA, are approximately 1350 nt in length (Approximately half that of the genome of these helper viruses) and, like the ToLCV-sat, are unrelated in sequence to helper viruses as well as requiring helper viruses for replication and movement in plants and insect transmission. Unlike the ToLCV-sat, P-DNA satellite affect the replication of their helper begomoviruses and alter the symptoms induced in some host plants. (Saunders et al, 2000, Briddon et al, 2001).

The majority of begomoviruses have a genome comprising two similar sized DNA components (DNA-A and DNA-B). The DNA-A component encodes a replication - associated protein (Rep) that is essential for viral DNA replication, a replication enhancer protein (REn), the coat protein (CP) and a transcription activator protein (TrAP) that controls late gene expression. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), both of which are essential for systemic infection of plants (Hanley-Bowdoin et al. 1999; Gafni and Epel, 2002). In contrast, some begomoviruses have only a single genomic component which resembles DNA-A, such as isolate of Tomato yellow leaf curl virus (TYLCV), Tomato leaf curl virus (TLCV), Ageratum yellow vein virus (AYVV) and Cotton leaf curl virus (CLCuV) (Kheyr-Pour et al, 1991; Navot et al, 1991. Dry e/ al, 1993; Tan et al, 1995; Briddon e^ a/., 2000).

Diseased Ageratum conyzoides plants contain a satellite DNA, referred to as DNA-P . AYVV and DNA P together from a disease complex that is responsible for the yellow vein phenotype (Saunders et al, 2000). A part from the sequence TAATATTAC, common to all geminiviruses and containing the initiation sites of rolling circle replication, DNA P shows negligible sequence homology either to AYVV DNA-A or to DNA-B components associated with other begomoviruses. Review of Literature 11

DNA-p depends on AYVV for replication and is encapsidated by AYVV- encoded coat protein. Systemic infection of A. conyzoides with AYVV alone is asymptomatic and viral DNA accumulation is reduced to 5% or less of that in the presence of DNA-p. Similarly, Cotton leaf curl disease (CLCuD) in Pakistan is caused by CLCuV and an associated DNA-p component (Briddon et al., 2001). More recently, (Mansoor et al. 2001) reported the association of a DNA-p component with Okra leaf curl disease in Pakistan. Plants affected by Ageratum yellow vein disease (AYVD) and CLCuD also contain small autonomously replicating nanovirus like components, called DNAl and DNA2, in addition to DNA-P and begomovirus components (Monsoor et al, 1999; Saunders et al, 2002a).

Geminivirus Genome

Four different genera are currently recognized, depending on differences in the genetic organization of the genome, host range and insect vector (Van Regerunortel et al, 2000, Fauquit & Stanley 2003, Fauquit et al, 2003).

1. Mastrevirus (such as Maize streak virus (MSV) and Wheat dwarf virus (WDV) are transmitted by leaf hopper, have a monopartite genome and most infect monocotyledonous species in the family Poaceae, but two species Bean yellow dwarf virus (BeYDV) (Liu et al, 1997) from South Africa and Tobacco yellow dwarf virus (TYDV) (Morris et al, 1992) from Australia are known to infect dicots also. Chickpea chlorotic dwarf virus (CPCDV) from India is transmitted by leaf hopper and is considered tentative to be a mastrevirus species (Horn et al, 1993), and one of the three commercially important plant virus diseases in Africa. Other mastreviruses occur in Asia, Australia and Pacific islands while Wheat dwarf virus (WDV) occur in Europe.

The genome of mastreviruses consist of four open reading frames (ORFs), two in virion sense (VI and V2) and two in complementary sense separated by a large (LIR) and a small (SIR) intergenic region located diagonally opposite to each other. ORFs C1 and C2 are spliced to produce Rep (Replication initiator protein). ORF CI encodes for RepA that activates virion expression, ORF VI encodes coat protein (CP) and ORF V2 encodes movement protein (MP). Mutational analysis of BYDV genes has shown that all Review of Literature 12

mastreviruses are similarly organized and have functionally equivalent genes irrespective of their origin from monocots or dicots (Liu et al., 1998). Mastrevirus have a single genomic DNA molecule of about 2600 nt which is transcribed bidirectionally. The Mastrevirus genome encodes four proteins two on the DNA strands i.e. contained in virus peirticle (the viral strand) and two on the complementary strand.

Mastrevirus genome contains a large (LIR) and a small (SIR) intergenic region, which are located at opposite sites of the circular viral genomic molecules are important for completing the DNA replication cycle (Fig 2.2) . Mastreviruses are characterized by- a. ~80nt long DNA sequence that is present in the viral particle, armealed to a part of SIR. b. The occurrence of splicing events in both the complementary sense (C-sense) and the viral sense (V-sense) transcripts.

The Mastrevirus genomes encode four proteins.

i) RepA protein, exclusive for this genome. ii) Rep protein, on the C-sense strand. iii) Movement protein (MP). iv) Coat protein on V-sense strand (Crisanto Gutierrez, 2002).

2. Curtovirus {Beet curly top virus, BCTV) transmitted by leafhopper, infect dicotyledenous plants and have a monopartite genome but its genetic organization differ from that of mastreviruses. They are mainly found in North America with one species in Turkey. BCTV was reported in late 19'^ century and infect sugar beet {Beta vulgaris) and also pepper, melons, beans, tomatoes, spinach and many ornamental plants (Varma and Malathi, 2003). Curtoviruses are transmitted in persistent circulative marmer mostly by beet leaf hopper {Eutettix tenellus) (Harrison and Ribinson, 2002).

In curtoviruses, three proteins are encoded in virion - sense - CP, MP and V2 protein which might control the balance between ssDNA and double stranded DNA (dsDNA) synthesis and these are four ORFs in complementary J. . .2M) S «U o >- 'Si) S oj O ^ B •s S «? . oy WD 7 V Oi ^U •^ r- 1 S Oi .2 > ^ M H M U U s "5 ... Q> > &£ a s « s S c o « — u tt = «4-i es

S k B« s<» u O K f ••s Pi '> 1 « l-N 'fn N CZ3 o U u <: Ml .2 s t DD U O V « >- a S '5 s 2 ° o ¥ b u OS w«) fs e i ••• s P^ 4j I.V WD C/5 W) uT b £ Review of Literature 13

strand (CI, C2, C3 and C4) arranged similar to begomovirus DNA A (Harrison and Robinson, 2002). (Fig 2.2).

3. Begomoviruses such as (Bean golden mosaic virus, BGMV), African cassava mosaics virus (ACMV). Tomato golden mosaic virus (TGMV), Squash leaf curl virus (SLCV), cause several of the worlds most economically important plant diseases. They infect dicots in tropical and temperate climates. The vector whitefly (Bemisia tabaci Gennadius) is a species complex which transmits begomoviruses in the persistent circulative manner. Begomoviruses mostly have bipartite genome, designated as DNA-A and DNA-B, each 2.5-3.0 kb in length. DNA-A encodes the CP and the V-sense strand and a C-sense strand, four proteins: Rep, TrAP (a transcriptional activator). REn and C4. DNA-B encodes protein directly involved in movement of viral DNA: NSP (Nuclear shuttle protein formerly called BRI or BVl) and MPB (Movement protein encoded in the DNA-B component, formerly known as BLl or BCl). However, some begomoviruses, such as Tomato yellow leaf curl virus have a monopartite genome that resembles DNA-A.(Fig 2.3).

4. Topocuviruses, {Tomato pseudo-curly top virus (TPCTV) is transmitted by tree hopper (Micrutalin malleifera) (Harrison and Robinson, 2002). It represents the least characterized genus of geminivirus. It has a monopartite genome and its genetic organization is similar to that of curtovirus. Topocuvirus have two ORFs in the virion sense and four in the complementary sense.(Fig 2.3).

The diseases caused by geminiviruses are easily recognized by their distinctive symptoms. Mastreviruses induce bright yellow streaks in the monocotyledenous hosts and generally yellowing and dwarfing in the dicotyledenous hosts while curtoviruses cause typical curly top symptoms in their hosts. Diverse symptoms develop in the plants infected with begomoviruses, which are broadly of three types.

(a) vein yellowing (b) vein mosaic and (c) leaf curl.

There has been a tremendous increase in the number of begomoviruses that have been isolated in recent years. In the early 1960 about 27 such viruses were known (Varma 1963), now more than 100 begomoviruses have been characterized (Fauquit et al., 2003), and a large number remain to be classified (Fauquit & Stanley, 2003). > o

> u o

'a s o u M s

•> o S o a61 ) n a s o u M S Review of Literature 14

During the last two decades a large number of new begomoviruses causing mosaic and leaf curl symptoms have emerged in various part of the world (Varma, 1990; Polston and Anderson, 1997; Padidam et al., 1999; Rybicki and Pieterson, 1999). The new mastreviruses and curtoviruses have also emerged but to a lesser extent.

The Geminivirus DNA replication cycle

After the viral genome reaches the nucleus, the first stage is the conversion of the genomic ssDNA into a super coiled covalently closed circular (ccc) dsDNA intermediate. The second stage leads to the amplification of viral ssDNA through a rolling cycle replication (RCR) mechanism. The final stage, which does not involve DNA replication events, include both the transport of genomic ssDNA to neighbouring cells and its encapsidation to form mature viral particles.

(1) ssDNA fc- ccc dsDNA (occurs in absence of viral protein). RCR (2) ssDNA • amplification

A characteristic feature of geminiviruses is the presence of a sequence in their intergenic region that contains an invariant 9 nt sequence (TAATATT I AC), where the arrow marks the site of initiation of DNA replication, mapped both in vivo and in vitro. This conserved sequence is crucial for viral DNA replication and most likely is part, at some stage, of a stem - loop structure required for initiation. Initiation of DNA replication depends on the specific interaction of the Rep protein with its cognate binding site(s) within the replicator sequence.

Genomic organization of begomoviruses

Begomovirus have either bipartite or monopartite genome. Begomovirus originating in the New World have a bipartite genome organization whereas those from the Old World have either bipartite or monopartite genomes (Brown et al, 2002). In monopartite begomovirus such as Tomato yellow leaf curl viruses from Israel (Navot et al., 1991) and Sardinia (Kheyr-Pour et al., 1991), only a single component similar to DNA-A of bipartite begomoviruses in genome organization has been identified and shown to be enough for producing infectivity when reintroduced in tomato fiilfilling Koch's postulate and confirming that the single genomic component is solely responsible for disease development. Review of Literature 15

Recently, a satellite molecule has also been found associated with monopartite begomovirus. These satellite molecules called as DNA P have been found to give rise to typical disease symptoms when infecting along with their respective DNA-A monopartite component in begomoviruses like Ageratum yellow vein virus (AYVV) (Saunders et al., 2000), Bhendi yellow vein mosaic virus (BYVMV) (Jose and Usha, 2003). In DNA p, only a single positionally conversed ORF had been identified, the CI gene (Briddon et al., 2003), but its function remains unclear, though it has the ability to suppress gene silencing when transiently expressed in Nicotiana benthamiana (Mansoor et al., unpublished). Species in the genus begomovirus (type species Bean golden mosaic v/rw.s'-Puerto Rico) infect dicots and are transmitted in the persistent circulative manner by the whitefly Bemisia tabaci. B. tabaci occurs world wide in the tropical, subtropical and warm temprate regions and has evolved into different biotypes in different areas (Perrin TR, 2001). The disease caused by begomovirus result in yield losses valued at billion of pounds sterling per armum (Harrison et al, 2002). In begomovirus, DNA-A and DNA-B component constitute bipartite genome. DNA-A is essential for replication and encapsidation (Rogers et al., 1986; Townsend et al., 1986; Sunter et al, 1987) and DNA- B play a role in systematic movement and symptoms production (Etessami et al., 1988; Noueiry et al, 1994). DNA-A of all begomovirus has five ORFs, of which one (AVI, also called ARl) is on the virion DNA strand and the other four (ACl, AC2, AC3, and AC4 also called as ALl, AL2, AL3 and AL4 respectively) are on the complementary strand. The viral strand (ORFs) code for the coat protein and for a protein required for cell to cell movement of the virus. The protein encoded by the ORFs on the complementary strand are both involved in viral DNA replication, and are translated from spliced and unspliced version of the same mRNA (Harrison et al, 2002).

Begomoviruses from the Old World possess an additional ORFs (AV2), not found in New World begomovirus (encyclopedia of virology vol.1).

DNA-A has two ORFs in the virion sense [AVI/ALl-coat protein (CP) and AV2/AL2-pre coat protein] and found ORFs in the complementary sense [ACl/ALl- replication initiator protein (Rep), AC2/AL2-transcription activator protein (TrAP), AC3/AL3-replication enhancer protein (REn), AC4/AL4 and AC5/AL5]. DNA-B has Review of Literature 16

one ORF each in virion strand [BVl/BRl-nuclear shuttle protein (NSP)] and complementary strand [BCl/BLl-movement protein (MP)].

Common region

DNA-A and DNA-B sequence are different from each other except for an approximately 200 bp intergenic region and hence that region is called as common region (CR). The CR is present in the intergenic region between ORFs AVI and ACl in DNA-A and between ORFs BVl and BCl in DNA-B and it is highly specific for a virus. CR is the only region with significant sequence similarity between DNA-A and DNA-B components of the same virus. The CR has many regulatory elements including two TATA motifs one for ORFs AV1/AV2 and another for ORF AC1/AC4. Apparently, the bi-directional promoters for these ORFs are also present in the CR region. CR also has binding site for Rep protein that are repeated (iterons), sometimes in perfectly and sometimes in inverted orientation (Fontes et al, 1994). Iterons sequences of different viruses vary in length, sequence, number and orientation. There are characteristic difference in the arrangements of the iterons between the Old World (Asia, Africa, Mediterranean and Australia) and the New World species (America) (Arguello - Astoroga et al., 1994) CR has the conserved nonanucleotide sequence present in all geminiviruses, TAATATTAC, with the eight nucleotide "A" serve as origin of replication (ori). The invariant nonanucleotide sequence present with in the loop of stem and loop structure with inverted and complementary stem sequence being variable in composition and length between different viruses (Harrison and Robinson, 1999).

Infectivity studies to demonstrate the bipartite or monopartite nature of the genome of geminiviruses

An important characteristic of the genome of a virus is to be able to replicate autonomously inside the infected plant cell. Only a complete uninterrupted genome will be able to infect. The ssDNA of geminiviruses is encapsidated inside the geminate particle. A number of geminiviruses have so far been characterized. They have either one or two genomic components. In case of geminiviruses having a single genomic component, systemic infection can be produced by introducing the intact genomic component. However, in case of viruses with two genomic components, systematic Review of Literature 17

infection results only after both intact genomes have been introduced. It is in this context that the infectivity studies are important, since these serve to demonstrate that the virus is mono or bi-genomic.

For geminiviruses, which were not transmitted mechanically. Agrobacterium mediated delivery was the method of choice. Agrobacterium mediated inoculation of plant viruses was first reported by Grimsley et al. 1987. It has been used to infect dicotyledonous plants with TGMV (Elmer et al, 1988a; Hayes et al, 1988a), ACMV (Morris et al, 1988) and monocotyledonous plants which are recalcitrant to mechanical inoculation, with Maize streak virus (MSV, Grimslay et al, 1987), Digitaria streak virus (DSV, Donson et al, 1988) and Wheat dwarf virus (WDV, Hayes et al, 1988b).

CLASSIFICATION OF GEMINIVIRUSES INTO SUBGROUP I, II, III AND IV Geminiviruses are transmitted in nature by insects and have been classified on the basis of the type of host they infect (monocot or dicot) and according to the vector responsible for their transmission (leaf hopper / treehopper or whiteflies) (Francki et al, 1991, Van Regenmortel et al, 1997). The traditional classification scheme has been shown in (Table 2.1).

Subgroup I: This subgroup includes viruses that have a monopartite genome, infect monocotyledons and are transmitted by leafhopper. The members are Maize streak virus (MSV, Howell 1984), Wheat dwarf virus (WDV, MacDowell et al, 1985) Digitaria streak virus (DSV, Donson et al, 1987) etc. (Table 2.1). Their genomes contain four ORFs and two non-coding intergenic region. These four ORFs diverge from the larger intergenic region and converge at the smaller intergenic region ORF C2 encoded by these viruses, does not contain an ATG initiation codon (Donson et al, 1987; Anderson et al, 1988; Briddon et al, 1992; Hughes et al, 1993). It has been shown that ORF C2 is a part of larger protein encoded by a spliced transcript (Accotto et al, 1989; Dekkar et al, 1991). Typical transcription initiation and termination sequences are located in the large and small intergenic regions, respectively. The large intergenic region also contains a hairpin structure, which includes the conserved sequence motifs 5'TAATATTAC-3' that is found in the common region of bipartite viruses (Fig. 2.2). Review of Literature \ g

Subgroup II This subgroup includes viruses which are also transmitted by leafhoppers but infect only dicots. This subgroup includes Beet curly top virus (BCTV) and perhaps Tomato pseudo curly top virus, a recently characterized geminivirus, transmitted by a treehopper (Briddon et al., 1996). The genome organization of BCTV (type member of the group) is different from the viruses belonging to subgroup I. The organization of ORFs is more similar to viruses belonging to subgroup III having four ORFs in the complementary sense (CI, C2, C3 and C4) and three in the virion sense (VI, V2 and V3) (Fig. 2.2). The organization however is not exactly similar as there are differences in the start and end points as well as in the size of the putative products.

Subgroup III Viruses belonging to this subgroup are all transmitted by whiteflies (called as whitefly transmitted geminiviruses, WTGs), infect dicots and have either bipartite or monopartite genome. Subgroup III viruses or WTGs are prevalent throughout the world and have been further divided into two subgroups Old World (OW) and New World (NW). Those belonging to the NW are all bipartite in nature while those belonging to the OW can have either bipartite or monopartite genome (Table 2.2). It describes the various members of this group while Fig. 2.3 shows the various ORFs encoded by their genome. The genome organization of DNA-A of WTGs from the NW is similar to those from the OW (DNA-A of bipartite or the only component of monopartite) except the AVl/Vl ORF (pre coat protein) which is absent from NW WTGs. Position of other ORFs encoded by their genomes are highly conserved (AVI, ACl, AC2, AC3 and AC4). The genome organization of DNA B of NW viruses is similar to that of the OW (bipartite viruses), with BVl and BCl being the ORFs in the virion and complementary sense, respectively.

Subgroup IV (Topocuviruses, {Tomato pseudo curly top virus). This subgroup has arisen from Beet Curly Top Virus,{BCTV type member of Curtovirus). According to ICTV 1999 (Faquet and Stanley, 2003), it is a separate group of geminivirus. Those viruses belonging to this group, are transmitted by treehopper (Micrutalin malleifera) (Harrison and Robinson, 2002). It has monopartite genome and its genetic organization is similar to that of curtovirus. Topocuvirus have two ORFs in the virion sense and four in the complementary sense (Fig. 2.3). Review of Literature \ 9

GEMINIVIRAL PROTEIN FUNCTION

ORF AVI (Coat protein)

The most abundant protein in infected tissues is the coat protein, encoded by 0RFV2 in leafhopper transmitted viruses of subgroup I (Morris et al, 1985) and by ORf AVI in bipartite geminiviruses (Kallender et al., 1988). The capsid, which is common in all the whitefly-transmitted geminiviruses, has one or more antigenic epitopes. It has been suggested that these may be determinants of vector specificity, which would indicate that the coat protein plays a predominant role in transmission of virus, by the vector (Roberts et al, 1984). Even in case of some Luteo-viruses similar observations were made (Gildow and Rochow, 1980; Gildow, 1982; Gildow, 1985). Geminiviruses are transmitted in a circulative manner (Harrison, 1985; Hull, 1994) and do not replicate in their insect vectors (Baulton and Markham, 1986), but interact specifically with the insect vectors (Cohen et al., 1989). It was shown that mutants of ACMV that do not produce coat protein were not acquired by the whitefly vector (Briddon et al, 1990). Role of the coat protein in determining insect- vector specificity was demonstrated recently by construction of a chimeric virus in which the coat protein gene of whitefly transmitted ACMV was replaced by that of the leafliopper transmitted BCTV (Briddon et al., 1990). The resuhing chimeric virus was thus transformed to a leafhopper-transmitted type and could be transmitted by leafhopper. It has been suggested that the epitopes on the coat protein interact with the insect gut wall membrane to enable virus mobilization into the hemocoel. The coat protein thus, serves to protect the viral nucleic acids and to provide vector specificity.

The requirement of coat protein for other fianctions, such as movement varies with different geminiviruses. It has been functionally proved that in case of leafhopper transmitted geminiviruses with monopartite genome coat protein (CP) is essential for spread of virus and development of symptoms. Mutations in CP prevent systemic infection of the host without affecting replication in protoplasts (Baulton et al, 1989; Briddon et al, 1989; Lazarowitz et al, 1989). This observation extends to whitefly transmitted geminiviruses inspite of limited similarities between their CP sequences (Howarth and Vandermark, 1989). This is however the limitation with WTGS having monopartite genomes, for example TLCVA (Australian strain of TLCV, Ridgen et al, 1993; Wartig et al, 1997). With WTGS having bipartite genomes, CP is not essential Review of Literature 20

for replication, spread and symptoms development (Stanley and Thownsend, 1986; Gardiner et al., 1988; Padidam et ai, 1995a).

CP determines transmission by an insect vector. For BGMV, mutation in CP resulted in loss of acquisition and transmission by Bemisia (abaci (Azzam et al., 1994). In Abutilon mosaic virus (AbMV), exchange of three amino acid in the CP of an otherwise non-transmissible isolate resulted in efficient whitefly transmission (Hohnle et al., 200\).

In bipartite begomoviruses, CP was not needed for mechanical inoculation or agroinoculation of a well-adapted host but was needed for whitefly transmission or for agroinoculation in a sub optimal host (Pooma et al., 1996). For monopartite begomoviruses like Tomato yellow leaf curl virus (TYLCV), CP was required for infectivity (Rigden et al., 1993). In addition to nuclear import, TYLCV CP had been foimd to play a role in nuclear export acting as a homologue to the bipartite begomoviruses BVl protein (Rojas et al., 2001). CP mutants accumulated wild type levels of dsDNA but only small amount of ssDNA suggesting that CP might play a role in sequestering ssDNA (Padidam et al., 1996). The requirement for the CP for efficient long distance movement may reflect its participation in vascular spread. This might ensure the availability of virions in the phloem for acquisition by insect vectors. This will be consistent with the data which suggests that the CP plays an essential role in virus acquisition and /or transmission by insect vectors (Briddon et al., 1990; Azzam et al., 1994).

ORF AV2 Pre coat protein

Only Old World begomoviruses found to possess ORF AV2 for a bipartite Tomato leaf curl virus from India. ORF AV2 had been found to play a role in virus movement (Padidam et al., 1996). It is speculated that for monopartite begomoviruses found only in the Old World, ORF AV2 is complementary, the function of virus movement rendering DNA-B redundant (Harrison and Robinson, 1999).

ORF ACl (Replication initiator protein) and ORF AC3 (Replication enhancer protein) Through mutational studies, Elmer et ai, 1988b have shown that the product of the AC 1 gene is necessary for replication. Similar results have been provided by (Hayes Review of Literature 21

and Buck 1989) and (Hanley Bowdoin et al., 1990), where they expressed the product of the ACI gene in transgenic plants and analyzed its ability to support replication of the DNA-B component of TGMV. Both the groups independently reached to the conclusion that the product of the ACI gene alone is sufficient for the replication process of the viral genome. Geminivirus replication entirely relies on the DNA replication enzymes of the host plant. Replication enhancer protein (REn) enhances viral DNA accumulation. This depends on interaction of REn with Rep, which would bind simultaneously to the stem-loop structure and to the upstream Rep-DNA complex (Henley - Bowdoin et al., 1996). There is no direct evidence for this model but it would serve to direct Rep-DNA complex to nicking site. Mutation in REn produced delayed onset and attenuation of symptoms (Sunter et al., 1990).

Rep protein

During replication, Rep specifically recognized the viral origin (Pontes et al., 1994), binds to specific sequence (repeats / iterons) found in the CR and cleaves the phosphodiester bond between the seventh and eight residues of the conserved nanomer 5TAATATTAC3' (Stanley, 1995). Rep remain bound covalently to the 5'-phosphate end and the 3'-hydroxyl end becomes available for rolling circle replication. After one replication cycle, Rep cleaves once again at the newly generated origin sequence. The Rep ligates the nascent 3' end of DNA with the previously generated 5' end releasing a unit genome length, circular ssDNA molecule (Bisaro, 1996). Thus, Rep act as an endonuclease and ligase to initiate and terminate rolling circle replication (Laufs et al., 1995b; Orozco and Hanley - Bowdin, 1998). The Rep proteins of all geminiviruses have an NTP-binding domain (Hanley Bowdoin et al., 1999) and the Rep proteins of TGMV and TYLCV are known to hydrolyze ATP (Orozco et al., 1997; Desbiez et al., 1995). Gorbalenya and Koonin (1993) proposed that Rep might function as a DNA helicase during replication. Rep acts as a transcriptional regulator to reports its own synthesis (Sunter et al., 1993; Eagle et al., 1994). Rep has also been shown to oligomerise with itself as well as with REn protein (Settlage et al., 1996).

DNA binding, cleaving and ligation domain of Rep is at N'-terminus, oligomerization domain is located almost in the middle and ATP hydrolyzing activity at C'-terminus (Orozco et al., 1997). Sequence analysis of rolling circle initiator proteins of eubacteria, eukaryotes and archaebacteria revealed three conserved motifs -1 Review of Literature 22

(FLTYP), II (HxH) and III (YxxK) (Ilyina and Koonin, 1992). Motif III with a tyrosin (Y) residue is involved in the nucleophillic attack of phosphodiester bond of DNA at ori and represents the active site of Rep for DNA cleavage (Laufs et ah, 1995a). Mutation of motifs I 11 and III of TGMV rep protein also blocked DNA cleavage and replication (Orozco and Henley Bowdoin, 1998). Two a-helices in the N'-terminus of Rep protein, which overlap DNA binding and cleavage domains, were conserved among different geminiviruses (Orozco et al., 1997). Mutation in these helices inhibited DNA binding and cleavage in vitro and viral replication in vivo, thereby proving that these helices are essential for Rep function (Orozco and Hanley - Bowdoin, 1998). For TYLCV Rep, the structure of the N'-terminus catalytic domain (4-21 amino acid) had been elucidated by nuclear magnetic resonance (NMR) spectroscopic studies. In TGMV Rep, another set of a-helices was found between amino acid 131-152. Mutation in these helices, through hampered oligomerization of Rep, downstream sequences were found more important for complex formation (Hanley - Bowdoin et al., 1999). ATPase domain of Rep found in the C-terminus was mapped between amino acid 182- 352 (Orozco et al, 1999). This region has a conserved motif - GxxxxGKT that corresponds to the P loop of the phosphate-binding site of NTP hydrolyzing proteins (Gorbalenya and Koonin, 1989). Mutation in this motif of TYLCV Rep and BGMV Rep interfered with the virus replication (Desbiez et al, 1995; Hanson et al, 1995).

Biochemical studies have revealed that Rep protein of TYLCVS and WDV possess a DNA cleavage activity specific for the replication origin (Heyraud-Nitschke et al., 1995; Laufs et al., 1995b). "Tyrosine" at 103 position of the rep proteins (which is conserved in all geminivirus Rep proteins) has been implicated and proven to be essential for cleavage of DNA at the origin (Stanley 1995; Laufs et al, 1995a). These workers independently analyzed the process of the cleavage activity by the replication protein of ACMV and TYLCVS, respectively. Both proved that the cleavage activity of the Rep protein works in the loop of the stem loop structure of the two geminiviruses and that the results for these two viruses can be generalized for all the geminiviruses. By in vitro studies they proved that the Rep protein specifically cleaves the viral origin.

Rep proteins also possess a DNA joining activity that permits the transfer of the 5' terminal phosphate of an oligonucleotide linked to the 3'-0H end of an Review of Literature 23

oligonucleotide recognized by Rep. In vitro none of these reactions catalyzed by the Rep proteins require ATP as an energy donor.

Replications

Gene ACl, located in the complementary strand of the DNA-A of bipartite geminiviruses is essential for viral replication in the infected leaves (Brough et al, 1988; Elmer et al, 1988b; Etessami et al, 1991). The small size of the geminivirus DNA's, their single stranded nature and the presence of double stranded replicative intermediates in the infected leaves suggested that they might replicate by the rolling circle replication mechanism (Gilbert and Dresser, 1968; Davis et al, 1987). An evidence for the above hypothesis was provided by (Saunders et al, 1991).

ORF AC2 (Transcription activator protein, TrAP)

TrAP induces virion sense promoters in DNA-A as well as in DNA-B. In TrAP mutants of Tomato golden mosaic virus (TGMV), there was no accumulation of coat protein and no infectivity was achieved. Also ssDNA accumulation was impaired just like CP mutation (Sunter et al., 1990). It was concluded that TrAP transactivates virion sense expression-CP in DNA-A as well as ORF BVl in DNA-B at the level of transcription (Sunter and Bisaro, 1991, Sunter and Bisrao, 1992). Sunter et a/.(1994) showed that TrAP transactivation was non-specific. TGMV virion sense promotes were transactivated by TrAP protein from other begomoviruses like ACMV and Squash leaf curl virus (SLCV) but transactivation by TrAP homology in curtoviruses-C2 protein was not achieved. TrAP was shown to activate CP by two distinct mechanisms i.e. by activation in mesophyll cells and derepression in phloem cells (Sunter and Bisaro, 1997).

ORFs AC4 and ACS

ORF AC4 is present within Rep protein but in the different frame thus coding for a different protein. The role played by ORF AC4 in the virus infection could not be investigated for TGMV (Pooma and Petty: 1996) but later ORF AC4 was presumed to weakly suppress Rep gene expression (Groning et al., 1994) and the binding site for AC4 protein mediated repression did not involve Rep binding site and is distinct (Eagle and Hanley-Bowdoin, 1997). In Tomato leaf curl virus, ORF C4 is neither required for Review of Literature 24

the virus replication nor for systematic spread of the virus but is considered to be a determinant of symptom severity (Rigden et al., 1994).

In the case of monopartite Begomovirus, (TYLCV), it was presumed that ORP C4 might play a role in delivering viral DNA to the cell periphery and the plasmodesmata and serve as BCl protein homologue (Jupin et al., 1994; Rojas et al., 2001). ORF ACS codes for a protein for which the functional significance is yet to be ascertained. It has also been observed in some YMV isolates like Pepper huasteco virus (PHV) (Torres Paecco et al., 1993), BGMV and Potato yellow mosaic virus (PYMV).

ORFs BVl (Nuclear shuttle protein, NSP) and BCl (Movement protein, MP)

In general, for bipartite begomoviruses, ORF BVl function as a nuclear shuttle protein (NSP) i.e. in transporting the virus DNA between nucleus and cytoplasm while ORF BCl (Movement protein, MP) function in cell to cell movement of the viral DNA (Gafhi and Epel, 2002).

For Bean dwarf mosaic virus (BDMV), Abutilon mosaic virus (ABMV) and SLCV, NSP and MP have been shown to function co-operatively in cell to cell movement (Sanderfoot et al, 1996, Zhang et al., 2001). For SLCV, it was suggested that NSP was involved in shuttling viral ssDNA both into and out of the nucleus (Pascal et al, 1994). The BVl-ssDNA complex is trapped by BCl and BCl: BVl ssDNA complex is directed to the cell periphery (Sanderfoot and Lazarowitz, 1996).

Studies with ACMV did not support the suggestion that NSP and MP function in concert in cell to cell spread. It was suggested that NSP supported local spread while MP, possibly along with NSP, aided in systemic spread (Von Amim et al., 1993).

MP has been shown to be the determinant of pathogenecity (Symptom-inducing element) of bipartite begomoviruses (Von Amim and Stanley, 1992). The viral disease like phenotype seen in transgenic plants expressing MP might be due to interference of MP with macromolecular intercellular trafficking and 3' terminus of MP was associated with symptom development (Pascal et al, 1993).

In order to cause disease, a plant virus must move out of the original inoculated cell and spread systemically through the host plant. The functions associated with movement of the virus and its systemic spread are encoded by the viral genome. In case Review of Literature 25

of geminiviruses also, which have either monopartite or bipartite genomes, sequences encoding viral movement function have been identified. In the case of bipartite geminivirus, it has been found that DNA-B is essential for systemic movement of the virus. On the other hand monopartite viruses are dependent only on the genes (the proteins encoded by these genes) of DNA-A for its movement, since another genomic component is missing. Phloem limited viruses (geminiviruses and luteoviruses) are transmitted by insect vectors. These are directly introduced into the sieve element (SE) as virions, during insect feeding. Once within the translocation system, the virus must exit the SE into phloem - associated cells for replication, (Lucas and Gilbertson, 1994). Since geminiviruses replicate in the nucleus, there is a need to spread the infection from nucleus to the cytoplasm and from there to the cell periphery and to other cells.

Rolling circle replication

Geminiviruses replicate via rolling circle replication (RCR) in analogy to T4 bacteriphage replication, which also have single-stranded circular DNA. For ACMV, two-dimensional electrophoresis to examine the replicative intermediates generated during infection, which established the rolling circle mode of replication for geminiviruses (Saunders et al., 1991). The next stage, the rolling circle phase, requires the concerted action of viral Rep protein and other viral proteins with cellular factors and leads to the production of new dsDNA and ssDNA viral forms, (Stenger et ai, 1991; Stanley, 1995). The double-stranded replicative form serves as a template for transcription as well as ssDNA synthesis. Single-stranded DNA a) enters replicative cycle or b) gets transported from cell-to-cell by viral movement protein or c) gets encapsidated by viral coat protein (Gutierrez, 2000).

Transcription regulation

Rep is known to auto-regulate its own expression. The Rep binding interon is located between TATA box and Rep transcription start site and when Rep binds to the interon, it down-regulates its own expression (Sunter et al., 1993; Eagle et ai, 1994; Groning et al., 1994). The C-terminus domain of mastrevirus Rep has a transactivation domain (Horwath et al., 1998), which might be responsible for its transcriptional regulation. For begomoviruses too, the same domain was expected to be responsible for Rep auto-regulation (Gutierrez, 2002). Auto-regulation does not require a fiinctional Review of Literature 26

viral ori suggesting that Rep binding site acts independently during transcription and replication (Eagle et al., 1994). In TGMV, AL4 mediated repression of ALl promoter has been reported which does not involve ALl binding site. Also, a TATA box element located immediately upstream of the Rep binding site and a G-box element located at the base of stem -loop structure is involved in activation of ALl promoter (Eagle and Hanley-Bowden, 1997). Mutations in TATA-box and G-box motifs were detrimental to Rep promoter function. In particular, G-box mutants displayed very low activity indicating that this element is the primary Rep transcriptional activating sequence. TATA-box and G-box element share a role in replication as well as transcription. A host G-box transcription factor might activate Rep expression and facilitate Rep recruitment and binding to the origin, modulate chromatin assembly and origin accessibility or stabilize an origin conformation required for efficient replication (Hanley-Bowdoin et al., 1999). In ACMV, a domain responsible for negative regulation of Rep promoter by its product had been mapped to a 92 bp fragment located immediately upstream of Rep initiation codon encompassing the consensus TATA box and transcription start site (Hong and Stanley, 1995). Like TGMV, ACMV Rep protein could not fully silence its promoter. Though the equivalent sequences are involved in transcriptional regulation of Rep for both ACMV and TGMV, there are some differences too (Hong and Stanley, 1995). ACMV Rep promoter does not have a G-box factor and transcriptional regulation is regulated by multiple c/^'-elements. Also AC4 protein in ACMV did not negatively regulate Rep expression.

The virion sense promoters for DNA-A (CP) as well as DNA-B (NSP) in TGMV and ACMV have been mapped. They comprise the CR and downstream sequences that contain TATA box and transcription start site and were enough for virion sense gene activation when TrAP protein was supplied in trans (Sunter et al., 1990; Sunter and Bisaro, 1991; Sunter and Bisrao, 1992; Groning et al., 1994). Activation of virion sense promoter have been found to occur at the level of transcription (Sunter and Bisaro, 1992). AC2 regulation of virion sense promoter is non-specific, as it has been shown that TGMV DNA-A could complement non- infections ACMV and PYMV TrAP mutants in plants (Sung and Coutts, 1995a). This suggests that TrAP functions either through conserved DNA sequences or through conserved interaction with host factors or both. Within virion sense promoters, a Review of Literature 27

conserved sequence called the conserved late element (CLE), although not ubiquitous, is present in most of the begomoviruses and has been implicated for TrAP interaction (Arguello-Astorga et al., 1994). TrAP was found to induce C? expression in TGMV by activating it in mesophyll cells and derepressing it in phloem cells (Sunter and Bisaro, 1997). Also Rep did not regulate MP transcription (Sunter et al., 1993), indicating that MP has different promoter sequences from that of Rep. Thus, in contrast to virion sense expression, where both genomic components have similar regulation, complementary sense expression is distinct for DNA- A and DNA-B.

Thus, in geminivirus transcription. Rep is active in early phase of viral life cycle. When sufficient Rep and AC4 protein accumulates, these promoters are repressed may be in order to limit their interference with the function of downstream TrAP promoters. TrAP and MP promoters are activated midway in viral life cycle. TrAP protein regulates virion sense gene expression and thus CP and NSP are late genes produced late in the viral infectivity cycle. This expression strategy was proposed by Howarth et al. 1985, but fiirther detailed studies are required to establish this hypothesis.

Phylogeny of begomoviruses

For begomoviruses, DNA-A and their conserved protein products CP and Rep are used for phylogenetic analysis. DNA-B sequences are more diverse than DNA-A and there are fewer conserved elements in DNA-B and hence, in general thay are not used for phylogenetic analysis (Harrison and Robinson, 1999). When CP and Rep protein of different begomoviruses were compared, based on geographical origins, they were divided into the Old World (Asian, African, Mediterranean and Australian) and the New World (America) viruses (Howarth and Vandemark, 1989). This kind of geography-related lineage was confirmed by further analysis including more begomoviruses (Hong and Harrison, 1995; Rybicki, 1994). Within the Old Word branch, three subclusters were discerned for CP: one with viruses from African and Mediterranean region, second with Asian viruses and third having viruses from China and Australia (Harrison and Robinson, 1999). The Old Word and the New World division of begomoviruses was further established when complete nucleotide sequence of DNA-A of 120 geminivirus species were analysed by Fauquet and Stanley, 2003. Currently, seven different virus clusters were discerned for begomoviruses-New World Review of Literature 28

virases comprising meso and Latin American viruses and viruses infecting sweet potato (sweepo viruses): Old World viruses comprising African, Indian, Asian viruses and viruses infecting legumes in India and Africa (legumoviruses) (Fauquet and Stanley, 2003). The phylogeny of geminiviruses is extremely stable, even when the viruses used for comparison were increased from 36 in 1989 to 277 in 2002. This aspect of stability in the phylogeny of geminiviruses would help in elucidating their evolution.

Viruses infecting Calendula

Vela (1972) isolated a virus from Calendula officinalis L. producing characteristic mosaic symptoms on Tobacco and N. glutinosa. Elongated particles 15x730-750m^ and amorphous inclusion bodies were observed. The distribution pattern of these was similar to those induced by the Potato Virus Ygp, but tubules similar to the once found in X-bodies from Tobacco mosaic virus were also seen.

In her paper (Padma et al, 1975) reported certain observation on the occurrence of mosaic disease on Calendula officinalis L,. In her report, she said that the disease is recorded for the first time from India and was sap transmissible on the A^. glutinosa and Chenopodium amaranticolor. The disease was also transmitted by grafting from calendula to calendula and also by Aphis gossypii and Myzus persicae.

Virus disease showing yellow net symptoms on Calendula officinalis was observed in Kasimpur, Aligarh by Naqvi et al, 1989. The virus was found to be mechanically transmitted from infected to healthy young seedlings of C. officinalis. In extract of C. officinalis leaves, the virus isolate lost its infectivity after 10 min of exposure at 65 °C but not at 60 °C and after dilution to 10"^ but not to 10"^. The virus was able to retain its infectivity upto 76h, when stored at room temperature (25+5°C). The virus was transmitted by two species of aphids viz. A. gossypii Glove, and M persicae Sulz in non persistent manner. The causal virus of Calendula yellow net disease was suspected to belong to cucumovirus group.

Purification and properties of Calendula yellow net virus was performed by Naqvi and Samad, 1985. The virus, causing yellow net of foliage and stunting in Calendula officinalis L. was purified by extraction with 0.05 M phosphate buffer pH 7.5 and chloroform followed by 3 cycles of differential centrifugation. Purified preparation was infectious and had a typical nucleoprotein spectrum. The virus Review of Literature 29

sedimented as a single band in sucrose density gradient columns and had 1 centrifugal component with a sedimentation coefficient of 86S. Particles were spherical, and of C. 35 nm diameter. RNA, isolated from purified virus preparation was found infectious. It was concluded that the virus was similar to Cucumber mosaic virus.

Hristova, et al, 1994 reported that indistinct mosaic symptoms were found on green house grown C. officinalis near the town of Pravets, Bulgaria. Based on host rang studies, serological and EM examination, the virus isolate from C. offiicinalis was identified as Tobacco mosaic tobamovirus.

Gupta and Verma (1983) describe a calendula rosette disease. The disease described and thought to be caused by virus affected 10-30% of C. offiicinalis surveyed in Delhi. It was transmitted by grafting and by whitefly (Bemisia (abaci), but not by sap extracted from affected leaves or by aphids. The symptoms, host range and transmission differs from those previously reported virus diseases of C. offiicinalis.

Lisa et al. (1979) observed C. offiicinalis, infected by Cucumber mosaic virus and Turnip mosaic virus, singly or together. The plant showed reduced growth, various degrees of leaf mosaic or mottling and fewer marketable flowers, but no visible flower symptoms.

Moreover, the calendula plant has also been found infected by Tomato chlorosis virus (ToCV) of the genus Crinivirus in the family Closteroviridae, which has broad host range as it includes 24 plant species in seven plant families. (Gail et al. 1999).

Khan et al. (2005) reported natural infection of begomovirus on Calendula offiicinalis L., plant showed yellowing of the vein and stunted growth of whole plant.

POLYMERASE CHAIN REACATION AS A TOOL TO ANALYZE VIRAL GENOME Polymerase chain reaction (PCR) is a technique for the in vitro amplification of specific DNA sequence by the simultaneous primer extension of the complementary strands of DNA. The PCR method was devised and named by Mullis and Faloona, 1987 although the principles had been described in detail by Khorana and colleagues (Kleppe et al.,\91\; Panet and Khorana, 1974) over a decade earlier. PCR is capable of producing a selective enrichment of a specific DNA sequence by a factor of 10^. The PCR thus provides a simple ingenious method to exponentially amplify specific DNA Review of Literature 30

sequences by in vitro DNA synthesis. PCR amplification requires two synthetic oligonucleotide primers that are complementary to the sequence flanking the DNA segment to be amplified. These primers define the ends of the DNA (target) that will be duplicated when a DNA sample is heated the two complementary strands separate, allowing the primers to bind to the flanking sequence, one on each strand. In the presence of dNTPs and a DNA polymerizing enzyme (which was originally the Klenow fragment of E. coli DNA Pol I), subsequently replaced by Taq DNA polymerase a heat stable DNA polymerase from the thermophilic bacterium {Thermus aquaticus), the primers then initiate the synthesis of two new strands, complementary to the original targets. Moreover, since the target product are also complementary to and capable of binding primers, each successive cycle essentially doubles the amount of DNA synthesized in the previous cycle. Accumulation of the specific target fragments is exponential, described by the reaction 2" where "n" is the number of cycles. The technique works well with degraded DNA, as well as with intact DNA and with pieces as small as 50-100 bp being amplified even when the DNA is not in a very pure form.

PCR offers several advantages compared to more traditional methods of diagnosis, organism need not be cultured prior to their detection by PCR, the technique possess exquisite sensitivity, with the theoretical potential to detect a single target molecule in a complex mixture without using radioactive probes and it is rapid and versatile. The technique may be used to investigate precise question about the composition of pathogen population and the genetic diversity of viruses (Gilberston et ah, 1991b; Robertson et ah, 1991). The use of PCR for diagnosis of plant diseases and other applications in plant pathology have been reviewed by Henson and French, 1993. Because PCR amplifies nucleic acid, the technique could be useful in overcoming many of the present difficulties associated with serological detection methods, for example low titer of antigen, cross reaction of antibodies with heterologous antigens and development and environmental regulation of antigen production. Furthermore, small amounts of plant samples, which can be fresh, frozen or even dried are suitable for PCR method, have been used for detection and to determine the genetic variability of plant viruses, including Luteoviruses (Robertson et al, 1991), Potyviruses (Vunsh et ai, 1990; Langeveld et al., 1991) and leafhopper transmitted geminiviruses infecting monocots (Rybicki and Hughes, 1990; Stenger and McMohan, 1997). Review of Literature 31

Detection and diagnosis of geminiviruses

In general, PCR is very useful in the detection and diagnosis of viruses, viroids and other plant pathogens. Because of the great sensitivity, the PCR provides a good alternative to other diagnostic methods and can speed diagnosis, reduce the sample size required and often eliminates the need for radioactive probes. Geminiviruses are well suited to detection and identification by PCR because they replicate via a double stranded, circular DNA intermediate the replicate form (Stanley, 1991) which can serve as a template for amplification by PCR. The genome of WTGs constitutes a number of regions which are highly conserved between viruses and hence can be used to design degenerate primers (Rojas et al., 1993, Tan et al., 1995). The highly conserved regions were identified for primers design by aligning derived amino acid and/or nucleotide sequences of at least 10 of the characterized WTGs from the Old as well as from the New World and determining consensus sequences. The conserved regions which are selected for primers design include:

1. The region in the ACl gene, which encodes for the conserved amino acid sequence the Try Gly Lys Thr Met Trp Ala (the putative NTP binding site). 2. The sequence nears the amino terminus of the coat protein, which encodes for the conserved amino acid sequence MetTyr ArgLysProArg. 3. The stem loop-region, which includes the conserved nonanucleotide, is conserved in all geminiviruses. 4. The sequences near the start site of the AC 1 gene. 5. The sequence nears the 3' end of the coat protein gene.

The primers mentioned above can be used to amplify any part of the genome and in various combinations (Gilbertson et al,. 1991b) demonstrated that PCR can rapidly and efficiently detect BGMV and could be useful in studies of variability and epidemiology of viruses. Detection of WTGs in plants and vector insects by PCR reaction using degenerate primers, designed for amplification of an approximately 500 bp fragment of DNA-A is also reported by Deng et al., 1994. A highly simplified PCR assay exclusively specific for subgroup III geminiviruses has been developed which permits the detection of a geographically diverse collection of WTGs infecting cultivated crops, ornamentals and weed hosts with minimal sample preparation (Wyatt and Brown 1996). They have designed degenerate primers to anneal to the most Review of Literature 32

conserved site within the most conserved gene (coat protein) of WTGs. The primer set which ampUfies 550 bp fragment was used because of their anticipated conservation and their apparently conserved nature. Designing of universal primers from the highly conserved ACl region for the PCR amplification of near full length (17 bases short) DNA-A of dicot infecting geminiviruses (Briddon and Markham, 1994) has helped to develop diagnostics against these viruses. No such primers have been reported from DNA-B. The degenerate primers of DNA-A have been found highly successful and a number of unknown geminiviruses have now been characterized using these primers (Guzman etal., 1997).

Detection and diagnosis of other plant viruses

The availability of nucleotide sequence of many plant pathogens has made possible the development of PCR assays for the detection and diagnosis of several viroids, viruses, and other pathogens. Because of its great sensitivity, the PCR provides a good alternative to other diagnostic methods and can speed diagnosis, reduce the sample size required, and often eliminates the need for radioactive probes. Detection of viroids, viruses, bacteria, phytoplasma, fiingi and nematodes by PCR has impacted diagnostic practices, epidemiological studies as well as studies of pathogen host vector interactions. Hadidi and Yang (1990) reported the detection of viroids by RT-PCR amplification, successfully utilized RT-PCR for the detection of plant RNA viruses and plant viral RNA satellites from infected tissue and predicted the application potential of PCR technology in the field of plant pathology. Subsequently, RT-PCR has shown its value in improving the detection of viroids by RT-PCR requires 1-100 pg of total nucleic acids from infected tissue and it is 10 to 100 fold more sensitive than viroid detection by hybridization and 2500 fold more sensitive than return gel electrophoresis analysis (Hadidi and Yang, 1990). The RT-PCR method has also been successfully utilized for the detection of Grapevine virus A (GVA) from infected grapevine tissue (Minafra et al., 1992) and Potato leaf roll luteovirus (PLRV) from infected potato tissue and viruliferous aphids (Hadidi et al., 1993b). RT-PCR was more sensitive than molecular hybridization or ELISA for the detection of either GVA or PLRV (Minafra et al., 1992; 1993a; Hadidi et al., 1993b). Other viruses reported to be detected by RT- PCR using viral specific non degenerate primers include Beet western yellows and Beet mild yellowing viruses (Jones et al., 1991), Tobacco rattle virus (Robinson, 1992), Review of Literature 33

different badnaviruses, including Banana streak, Rice tungro bacilliform and Sugarcane bacilliform (Olszewski and Lockhart 1992), Wheat soil borne mosaic virus (Pennington et al., 1992) and whitefly transmitted geminiviruses (Rojas et al., 1992, 1993). Degenerate oligonucleotide primers complementary to conserved genomic sequences shared by members of subgroup I geminiviruses. Maize streak virus and other geminiviruses, which include Maize streak virus and other geminiviruses of grasses and cereals, were successfully used for virus detection by PCR with a sensitivity approximately lO"* fold greater than that of ELISA (Rybicki and Hughes, 1990). The RT-PCR or PCR method has been successfully utilized to detect viroids or viruses from infected seeds (Hadidi et al., 1991, Kohen et al., 1992), fruits (Hadidi and Yang, 1990), flower parts (Jones et al., 1991; Kohnen et al., 1992; Frenkel et al., 1993), leaves (Hadidi et al., 1991; Kanemastu et al., 1994; Korschineck et al., 1992; Levy et al., 1991; Hadidi et al., 1992; Rezaian et al., 1992; Minafra et al., 1993b), bark (Hadidi and Yang, 1990; Hadidi et al., 1992; Yang et al, 1992b) roots (Hadidi et al, 1991, 1993a,b). Potato tubers, and viruliferous insects (Lee et al, 1993; Hadidi et al, 1993b).

Advantages and disadvantages of PCR

PCR allows for detection of low titer pathogens which include conventional detection methods such as ELISA or dot-blot hybridization, identification of unknown pathogens (Hadidi et al, 1995) detection of known pathogens that are currently detected by lengthy bioassays (Hadidi and Yang, 1990; Hadidi et al, 1992; Rezaian et al, 1992.; Minafra et al, 1992; Yang et al, 1992a; Minafra et al, 1993a and b), detection of multiple and uiu-elated pathogens in a single PCR reaction (Minafra et al, 1993a), identification of mixed infections or disease complexes (Hadidi et al, 1993b), rapid and sensitive evaluation of plants post-pathogen elimination therapy, evaluation of cross protection (classical or transgenic) (Scorza et al, 1993); determination of specific sequence information with or without cloning from crude total nucleic acids (Hadidi et al, 1991; Yang et al, 1992a; Minafra et al, 1993a; Zhu and Hadidi, 1993; Levy and Hadidi, 1994) and generation of pathogen specific clones without pathogen purification (Hayes and Buck, 1990; Hadidi et al, 1991; Yang et al, 1992a; Minafra et al, 1993a and b: Levy and Hadidi, 1994). This list does not describe the complete list of potential pathogen, pathogen vector, or pathogen-plant characteristics or interactions that can be examined by PCR. PCR is primer directed. Thus primers can be designed to Review of Literature 34

specifically amplify pathogen DNA or cDNA from heterogeneous samples. This obviates the need to purify the pathogen from infected plant tissue. PCR can be performed on very small biological samples, herbarium-preserved fungi, and can be used to analyze unculturable obligate plant parasites.

The major disadvantages include:

1. The initial expenses in PCR laboratory setup,

2. The requirement of trained personnel, 3. Cautious laboratory practices must be followed to prevent possible contamination from sample to sample,

4. Primers design requires some knowledge of the target sequence or a related sequence from published sequence data and finally,

5. For positive identification of specific target sequences it is useftil to obtain a specific or related clone for hybridization (this is especially important during the development of a new PCR assay when many bands may be generated prior to changes in PCR parameters) (Hadidi et ai, 1995). Materials and Methods MATERIALS AND METHODS

GENERAL PROCEDURES

Raising and maintenance of the host plants

Potting medium

Potting medium was prepared by mixing sand, soil and farmyard manure in a ratio of 1:1:1, clay pots and the medium were sterilized by autoclaving for 1 hr at 121°C and 15 lb per square inch.

Test Plants

For most of the purpose tomato, tobacco, chilli and calendula seedlings were raised in earthen pots of 30-cm in diameter and transplanted into clay pots of 15-cm in diameter. These plants were raised in an insect proof glass house with natural light and temperature conditions. However, during off season plants were grown at temperature ranging from 25-28''C in light and temperature controlled glass house.

Maintenance of virus culture

The culture of the virus was maintained on Calendula qfficinalisL.hy inoculating healthy seedlings at 3-4-leaf stage after every 30-45 days interval. In addition, on several occasions, twigs from infected plants were rooted in Hoagland solution and transferred into pots and into the green house.

Hoagland solution (Ix per liter) Ca (NO3)2-500 mg, KH2PO4-I25 mg

KNO3-I25 mg, Mg (804)2-125 mg, MS trace stock -1 ml and iron stock-1ml (Murashige and Skoog, 1962).

BIOLOGICAL CHARACTERIZATIONS

TRANSMISSION

Mechanical (Sap) transmission

For the transmission of Calendula yellow vein virus from naturally infected plant of Calendula, young leaves were macerated using a sterile mortor and pestle in O.IM potassium phosphate buffer (pH 7.0) containing 0.1% sodium sulphite and 5 mM EDTA. The slurry was squeezed through two folds of muslin cloth and the sap was Materials and Methods 36

rubbed onto carborundum-dusted leaves of Calendula and other seedlings using forefinger.

Aphid transmission

The aphid transmission tests were performed following the method of (Noordam, 1973).

Rearing of aphids

Aphids colonies were bread on appropriate plants as mentioned below.

Aphids species Host plant 1. Myzus persicae Nicotiana tabacum var. White Burley 2. Aphis gossypii Cucumis sativus 3. Brevicoryne brassicae Brassica oleracea var. capitata

Single adult apterous aphid of each species after starving for 6-7 hr was placed on a leaf and kept in a petridish on a moistened filter paper. Young nymphs produced were used for initiating colonies. Plants harbouring colonies were maintained in cages of 90x90x90 cm size. Fresh colonies were maintained by transferring them to fresh healthy plants after every 15 days intervals.

Testing for non-persistant transformation

Aphids were starved for 1-2 hr and then allowed for an acquisition access of 2 min on infected plants. Aphids were then transferred to different plant species (7 aphids / plant) and given an inoculation access of 2 hr before killing. The plants were kept under insect proof condition and observed for symptoms.

Testing for persistant transmission

Aphids were given an acquisition of 24 hr on infected calendula plant, then the aphids were moved to a series of different healthy plant species at 24 hr intervals. All the plants were kept under insect proof condition for 30-40 days and observed for symptoms (Systemic as well as local infection).

Whitefly transmission

Whitefly transmission test was performed as described by (Srivastava et al. \911). Materials and Methods "hi

Breeding virus free whiteflies

Since whiteflies are able to carry more than one virus at a time, whiteflies collected from Clitoria ternatia plants growing in the field were multiplied on Clitoria ternatia under controlled condition. Transfer of a generation of whiteflies to Clitoria plants made them virus free. None of the transmission test done with whiteflies grown on Clitoria ternatia produced any symptoms on test plant such as Nicotiana tabacum and Lycopersicon esculentum, (known hosts of most of the geminiviruses). These whiteflies were also tested by dot blot hybridization using general geminiviral probes for the presence or absence of the virus. Non-viruliferous whiteflies, thus multiplied were used in transmission studies.

Handling of whiteflies

Trapper

A piece of muslin cloth was tied on one end of a hollow glass tube of 15 cm length and 5 cm diameter. To trap the whiteflies, the open end of the glass tube was brought near the whitefly and air was sucked in through the covered end. In such a trapper approximately 100 whiteflies could be trapped at one time.

Acquisition and inoculation feeding cage

A plastic cage with a detachable lid at one end was used as the acquisition and feeding cage. On one end, a wire mesh (able to retains whiteflies) was fitted. A hole (2- 3 cm in diameter) was made in the center of the lid. At the time of using the cage, a conical flask was taken and was filled with either water or Hoagland solution. Lid of the container was placed on the flask and a twig of naturally infected calendula plant was passed through the opening in the lid so as to be dipping in water. Container having whiteflies was then made to cover the twig over the lid of the container already placed on the conical flask. After the feeding was over container was removed and the twig was tapped slowly to disturb the whiteflies, which then collected on the walls of the container. These viruliferous whiteflies in the container were given access to healthy calendula seedlings (inoculation access) at the 3-4 leaf stages. The container with whiteflies was placed over the twig of healthy calendula plant in the same way as was done initially to allow the whiteflies access to the diseased plants covering the Materials and Methods 38

container with a dark cloth or paper helped in forcing the whiteflies to settle and feed on the leaves of the seedlings.

Testing for transmission of the virus

Non-viruliferous 5-10 whiteflies were given an acquisition access period of 24 hr to the twig of calendula plant showing yellow vein symptoms, collected from the field. These whiteflies were transferred to healthy calendula seedlings at the 2-4 leaf stages and given an inoculation access period of 24 hr. After that, whiteflies were killed by spraying 0.1% (v/v) dimecron. Plants were kept under insect proof condition and observed for development of symptoms.

Seed transmission

Seed transmission test was also carried out to check out that the virus (isolated from calendula) is seed transmissible or not. For this, mature seeds were collected from naturally infected calendula plants and were sown in the sterilized soil filled pots for their germination. The pots were kept under insect proof condition in a greenhouse for the observation of disease symptoms upto their flowering stage.

Whitefly transmission of Calendula virus to other host plants

To check the host range of calendula yellow vein virus by whitefly transmission, several plant species viz. tobacco, chilli, tomato and datura were tested as in case of infected calendula to healthy seedlings of calendula.

MOLECULAR CHARACTERIZATION

Midi Method of plant DNA extraction by Dellaporta's (1983)

Plant DNA was extracted by the procedure detailed by Dellaporta et al., 1983. The tissue was frozen in liquid nitrogen and ground using a mortar and pestle to a fine powder, not allowing the tissue to thaw in between. All the powdered tissue was transferred to a centriftige tube containing 50 ml extraction buffer (for 5 g of leaf tissue) supplemented with 500 |il P-mercaptoethanol. The powdered tissue was suspended thoroughly by swirling slowly. This was followed by the addition of 3.3 ml of 20% SDS, mixed properly and incubated at 65°C for 15 min. After incubation, the content were cooled by incubating on ice for 15-20 min, followed by the addition of 17 ml of 5 M potassium acetate buffer (pH 5.5), mixed thoroughly and incubated on ice Materials and Methods 3 9

for 20 min. The debris were pelleted down by centriftigation at 10,000 rpm for 20 min using GSA rotor at 4°C and the supernatant was carefully decanted into a fresh tube, preferably filtered through two layers of muslin cloth. To the supernatant 33 ml of isopropanol was added, mixed and incubated at room temperature for 30 min for precipitating the nucleic acid.

The precipitated nucleic acid was collected by centrifugation at 10,000 rpm for 20 min in a GSA rotor at room temperature. Pellet of the nucleic acid was dried in the DNA drier and resuspended in TE50 buffer. The suspended nucleic acid was successively extracted with equal volume of phenol, phenol-chloroform and chloroform (chloroform is a 24:1 mixture of chloroform: isoamylalcohol) while Phenol is tris saturated unless otherwise stated. After centrifugation at 10,000 rpm in SS-34 rotor at room temperature for 10 min, the supernatant was collected, supplemented with 1/10* volume of 3 M sodium acetate (pH 4.8) and precipitated by 2.5 volume of absolute alcohol. After incubation at -20°C overnight, the precipitated nucleic acid was recovered by centrifugation of 12,500 rpm for 20 min in a SS-34 rotor at 4°C. Finally, the pellet was vaccuum dried and resuspended in appropriate volume of TE buffer.

Extraction buffer 100 mM Tris HCl (pH 8.0), 50 mM EDTA (pH 8.0) and 500 mM NaCl, 1 ml P-mercaptoethanol per 100 ml of extraction buffer to be added after autoclaving.

5 M Potassium acetate:- 49 g Potassium acetate per 100 ml solution.

TE50 50 mM Tris HCl (pH 8) and 1 mM EDTA (pH 8).

Mini DNA extraction method by Dellaportas (1983)

The Mini DNA extraction methods was used to isolate the DNA from other plants, used for host range studies.

1. Leaf tissue (lOOmg) was ground in liquid nitrogen using a mortar and pestle to a fine powder and transferred to microfuge tube.

2. Added 1ml, w/v extraction buffer supplemented with 1% P marcoptoethanol and 62.5^1 SDS (20%). The slurry was vigorously vortexed, incubated at 65 °C for 15 min and then cooled on ice. This was followed by addition of 1/3 volume 5M potassium acetate (pH5.5), mixed thoroughly, kept on ice for 15 min. Materials and Methods 40

3. The debris as precipitated by centrifliging at 10,000 rpm for 15 min at 4°C. The supernatant was carefully transferred to a fresh tube to which was added 600|al (0.6%) isopropanol, mixed and kept at RT for 30 min for precipitating nucleic acid. 4. The precipitated nucleic acid was collected by centrifliging at 10,000 rpm for 15 min at RT. The pellet was resuspended in 100^1 TE50 buffer. RNase was added to a final concentration of 20 ^ig/lml to the supernatant, mixed properly by gentle vortexing and incubated at 37°C for 30 min to remove all RNA. The nucleic acid was then extracted with equal volume of phenol: chloroform: isoamylalcohol (25:24:1).

5. The DNA was then precipitated with 2 volumes, v/v of absolute alcohol supplemented with I/IO"' volume 3 M sodium acetate, pH 5.2.

6. After keeping at -20°C overnight, the precipitated DNA was recovered by centrifiigation at 12,500 rpm for 20 min at 4°C, washed with 70% ethanol, dried in DNA drier and resuspended in sterile water.

Extraction buffer for total genomic DNA isolation from plant tissues 100 mM Tris (pH 8.0), 50 mM-EDTA, 500 mM NaCl.

TE50 50 mM Tris HCl pH 8.0,1.0 mM EDTA.

Tris-saturated phenol Extracted phenol with equal volume of 0.5 M Tris HCl (pH 8.0) thrice (pH of the supernatant should be (7.5-8.0) overlay phenol with 0.1 M Tris. HCl (pH 8.0) and add 0.1% p-ME.

Extraction of the plasmid DNA from the host bacterium

The procedure for plasmid DNA isolation by alkaline lysis method was as described by Bimboim and Doly (1979). Briefly.

1. 500 ml LB (Hi-media) containing 100 mg/L ampicillin and 12.5 mg/L tetracycline was inoculated with 25 ml of overnight grown culture (30 ml) ofE. coli (Strain DH5a and XLI Blue) with vigorous shaking till late log phase.

2. The culture was transferred to 3 tubes of GSA rotor of the Sorvall refrigerated centrifiige and centrifuged at 5,000 rpm for 15 min at 4°C. Materials and Methods 41

3. The bacterial cells were resuspended in 100 ml of ice cold STE and pelleted at 5000 rpm for 15 min at 4°C in 1 GSA centrifuge tube. 4. The cells were resuspended in 20 ml of ice-cold solution I and 2 ml of freshly prepared lysozyme was added to them. 5. This was followed by addition of 40 ml of freshly prepared solution II. The contents were mixed by gently inverting the tube several times and the tube was kept at RT for 10 min. 6. Following the addition of 20 ml of ice cold solution III, the tube was kept on ice for 10 min After centriftigation at 10,000 rpm for 15 min at 4°C, the supernatant was transferred to a fresh tube through 4 layers of cheese cloth. 7. DNA was precipitated with 0.6 volume (about 50 ml) isopropanol, mixed well and keeping at RT for 10 min. 8. The precipitated DNA was pelleted by centriftigation at 10,000 rpm for 15 min at RT. 9. The pellet was washed with 70% ethanol at RT, dried and finally dissolved in 3 ml T.E. buffer. 10. An aliquot of the DNA was checked for integrity by agarose gel electrophoresis and only the preparations with mostly supercoiled ccc forms were used according to (Sambrook et al., 1989).

STE 0.1 M Sodium chloride; 10 mM Tris.Cl,( pH 8.0); ImM EDTA, (pH 8.0).

Sol. I (GTE) 50 mM Glucose, 25 mM Tris.Cl,( pH 8.0); 10 mM EDTA,( pH 8.0).

Lysozyme 10 mg/ml in 10 mM Tris.CI,(pH 8.0).

Sol. II 0.2NNaOH;l%SDS.

Sol. Ill 5 M Potassium acetate 60 ml. Glacial acetic acid 11.5 ml, water 28.5 ml.

T.E. 10 mM Tris, HCl (pH 8.0) and 1 mM EDTA (pH 8.0).

Purification of plasmid DNA by polyethylene glycol (PEG) method

The method as described in (Sambrook et al., 1989) was essentially followed. Materials and Methods 42

The basic protocol involved alkaline lysis method as described above. After precipitation Wiih isopropanol, the plasmid DNA was processed in the following manner.

1. The nucleic acid solution (3 ml) was transferred to a centrifuge tube and 3 ml of ice-cold 5 M lithium chloride was added to it. 2. It was mixed properly, incubated on ice for 10 min and centrifuged at 10,000 rpm for 10 min at 4°C in SS34 rotor. The high molecular weight RNA pellet was discarded and the supernatant containing the plasmid DNA and the low molecular weight RNA was transferred to a fresh tube.

3. An equal volume of isopropanol was added, mixed thoroughly and the tube incubated at RT for 5 min. The nucleic acid was pelleted by centrifugation at 10,000 rpm for 10 min in SS34 rotor.

4. The supernatant was discarded and the pellet so obtained was washed with 70% alcohol, dried and resuspended in 500 |^1 T.E. (pH 8.0).

5. 0.5 jil (10 mg/ml stock) of DNAse free RNAse was added to it and incubated at 37°C for 30 min.

6. After the incubation, 500 ^1 of 1.6 M NaCl containing 13% (w/v) PEG 8,000 was added. The mix was incubated on ice for 15 min and centriftiged at 12,500 rpm for 10 min at 4°C. The supernatant was discarded and the pellet containing plasmid DNA was washed with 70% alcohol, dried and resuspended in 400 ^1 sterile water.

7. The DNA solution was successively extracted with phenol, phenol-chloroform and chloroform.

8. 100 ^1 of 10 M ammonium acetate was added to it and the DNA was precipitated with 2.5 volumes of absolute alcohol (v/v).

9. The mix was incubated on ice for 20 min, centriftiged at 12,500 rpm for 20 min at 4''C and the pellet obtained was washed with 80% ethanol twice, dried under vacuum drier and dissolved in appropriate volume of sterile water. The DNA concentration was determined by measuring the absorbance at 260 nm (A260 1.0=50 ^ig/ml DNA). Materials and Methods 43

Molecular cloning of PCR- product using (pGEM-T-Easy vector system-1, Promega corporation, USA)

Description of vector

The pGEM-T Easy Vector Systems are convenient systems for the cloning of PCR products. The vectors are prepared by cutting Promega's pGEM®-T Easy Vectors with EcoRV and adding a 3' terminal thymidine to both ends. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmids by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by certain thermostable polymerases. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3'-ends of the amplified fragments (3, 4).

The high copy number of pGEM®-T Easy Vectors contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the a-peptide coding region of the enzyme P-galactosidase. Insertional inactivation of the a-peptide allows recombinant clones to be directly identified by color screening on indicator plates. The multiple cloning region of the vector includes restriction sites conveniently arranged for use with Promega's Erase-a-Base®System for generating nested sets of deletions.

The pGEM-T Easy Vector contain multiple restriction sites within the multiple cloning region. These restriction sites allow for the release of the insert by digestion with a single restriction enzyme. The pGEM-T Easy Vector multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BsZI and Not I, thus providing three single-enzyme digestions for release of the insert, while the pGEM-T Vector cloning region is flanked by recognition sites for the enzyme BstZ I. Alternatively, a double-digestion may be used to release the insert from either vector (Fig 3.1 «& Fig 3.2)

The pGEM-T Easy Vector system include a 2X rapid ligation buffer for ligation of PCR product. Reaction using this buffer may be incubated for Ih at room temperature. The incubation period may be extended to increase the number of colonies after transformation. Generally, an over night incubation at 4°C will produce the maximum number transformant. c eg oo o VO m ON O 00 o r-- o T ~ 2: (N (N f^ •* u-1 VO 00

•~. a; -. N -^ -^ CL ,y ^ i-j to ts t ^ = to' "Jj ^ BQ Q, Co I CO oa ^ 'o 0^ V CI B a>

DS Ov Vl rN 0m0 CJN CM r~- O I •4-* 1 1 1 u r-~ o VO ck r^ oo VO t ON > (^^ m(N c^ R VO ON 0\ U O. u U C/5 C/) u 00 2 iii c u 60 1> E E (L> c c 3 E c _>, _>^ o CT 00 n _>. > o 'c o o 'ED o o a. ai O o a. a. o o o. H < < a o < i w O O "5 VO N U E 2 M CL. a. u D O 00 on D a. a. H c a. a < u< o u o. o u- u o u o o R s u ^ o cq u o u u o H < uU oO < H u O O H < < H o u H < U O O U o u < H OH < ^ ^- < o u < u o < I O u- < < < H < O U < H

00 < I U O O U < < H, < •4-» < H O U c o U O- ^ o u < o u o ^u: u o u m o u < < t: o u< u o < b: o u H < u o < u a o < H Hu o< B U O O o 2 < H cu H G O< o u U u< Ho < H H < U, < ^ t O U H < O u H < H < < o o < .^ >o c-) U O- § Materials and Methods 44

Protocols for Ligations Using the pGEM®-T Easy Vectors and the 2X Rapid Ligation Buffer

1. Briefly centrifuge the pGEM®-T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tubes. 2. Set up ligation reactions as described below. Note: Use 0.5 ml tubes known to have low DNA -binding capacity. 3. Vortex the 2X Rapid Ligation Buffer vigorously before each use. 4. Mix the reactions by pipetting. Incubate the reactions 1 hour at room temperature. Alternatively, if the maximum number of transformants is required, incubate the reactions overnight at 4°C. Optimizing Insert: Vector Molar Ratios

The pGEM-T Easy Vector Systems have been optimized using a I: I molar ratio of the Control Insert DNA to the vectors. However, ratios of 8:1 to 1:8 have been used successfully. If initial experiments with your PCR product are suboptimal, ratio optimization may be necessary. Ratios from 3:1 to 1:3 provide good initial parameters. The concentration of PCR product should be estimated by comparison to DNA mass standards on a gel or by using a fluorescent assay (7). The pGEM-T Easy Vectors are approximately 3 kb and are supplied at 50 ng/|il. To calculate the appropriate amount of PCR product (insert) to include in the ligation reaction, use the following equation. ng of vector x kb size of insert X insert: vector molar ratio=ng of insert Kb size of vector

For 3;]- Vector to insert ratio the amount of PCR-product required is calculated using the formula.

50 X 750 3 X = 37.5ng/|al 3000 1 Materials and Methods 45

Protocol for Ligation Using the PGEM-T-Easy vector and 2X Rapid Ligation Buffer

Ligation Reaction

2X Rapid Ligation Buffer, T4 DNA Ligase 5.0 |il pGEM-T Easy Vector (50ng) 1.0 \A PCR product (~38ng) 1.0 ^1 T4 DNA Ligase (3 Weiss unit/|al) 1.0 ^1 Deionized water to a final vol. 10.0 |il

PCR of coat protein region using specific primers (TLCV-CP) gene

Polymerase chain reaction (PCR) is a technique for the in vitro amplification of specific DNA sequence by the simultaneous primer extension of the complementary strand of DNA. PCR method was devised and named by Mullis and colleagues (Mullis and Faloona, 1987), although the principles had been described in detail by Khorana and colleagues (Kleppe et al, 1971, Panet and Khorana 1974).

PCR component for lOOjal reaction

DNA template (-lOOng) 5.0 \A Taq DNA polymerase (3U/^1) 1.0 |il Taq DNA polymerase (lOx buffer) 1 0.0 i^l dNTPs(lOmM) 2.0^1 Primer I (25 pmol) 2.0 |al Primer II (25 pmol) 2.0 fil MgCb (25 mM) 3.0 ^1 H20tomakeup 100.0 |al

PCR was performed using a pair of primers designed to the coat protein region of a well characterized begomovirus. Tomato leaf curl New Delhi Virus (ToLCNDV; Hallan, 1998).

Sequence TLCV-CPIT- 5' TTAATTTGTGACCGAATCAT 3'

Sequence TLCV-CPTI- 5' ATGGCGAAGCGACCAG 3' Materials and Methods 46

The PCR was conducted in a Gene Amp. 9700 (Perkin Elmer) thermal cycler. The PCR amplification parameters included initial template denaturation at 94°C for 5 min. This was followed by 30 cycles of PCR consisting of 30 sec. of denaturation at 94°C, 30 sec. of primer annealing at 47°C, and 40 sec. of primer extension at 72°C; and a final extension of 5 min at 72°C at the end of the cycles. The PCR-product so obtained was checked by electrophoresis on 1% agarose gel prepared in IXTAE buffer. Before carrying out ligation reaction, the PCR products were eluted from LMP agarose gel, ethanol precipitation at -20°C overnight, pelleted by centrifugation at 12,000 rpm for 15 min at 4°C, washed with 70% ethanol, vacuum dried and finally resuspended in sterile water. AGAROSE GEL ELECTROPHORESIS OF THE PLASMID AND TOTAL PLANT DNA

The gel electrophoresis was carried out using a submarine horizontal agarose slab gel as described by (Sambrook et al., 1989). Appropriate amount of agrose (0.8- 1.0% according to requirement) was dissolved in IXTAE or 0.5XTBE buffer by boiling to dissolve completely. This molten agarose was cooled to about 50°C and cast in an appropriate electrophoresis tray using a slot forming comb. After the agarose had solidified, the comb was removed and the gel was placed in the gel tank, containing IXTAE buffer for electrophoresis. The DNA was loaded in the wells of the gel after mixing with loading dye. Electrophoresis was carried out at a constant voltage of 5v/cm. After electrophoresis, the gel was stained with ethidium bromide (1.0 |ag/ml) and visualized under UV light.

Loading dye (6X) 15% FicoU 400, 0.25% bromophenol blue and 0.25% xylene cyanol-FF.

TBE buffer (lOX, per litre) 108 g Tris base, 55 g boric acid and 9.3 g EDTA.

TAE buffer (SOX per litre) 242 g Tris base, 57.1 ml glacial acetic acid and 100 ml EDTA (0.5 M pH 8.0). Materials and Methods 4 7

Detection of a satellite P-DNA molecule associated with Calendula isolate using universal primers

For the detection of association of a P-DNA satellite molecule with calendula isolate, p-DNA specific primers i.e. Beta IF (AGCCTTAGCTACGCCGGAGC) and Beta IR (GCTGCGTAGCGTAGAGGTTT) were used as a universal primers.

PCR amplification parameters included initial template denaturation at 94 °C for 5 min. This was followed by 30 cycle of PCR consisting of 1 min of denaturation at 94°C, 1 min of primers annealing at 55 °C, 1:30 min of primers extension at 72 °C, and a final extension of 7 min at the ends of the cycle. The PCR product thus obtained was checked by electrophoresis on 1% agarose gel.

PCR component for SOfil reaction

DNA Template (-lOOng) 3.0 ^1 Taq DNA polymerase (3U/^1) 1.0 |J,1 Taq DNA polymerase (IOXbuffer) 5.0 ^il dNTPs(lOmM) 1.0^1 Primer I (25pmol) 1.0 |il Primer II (25pmol) 1.0 ^il Mgcb (25pmol) 3.0 ^1 Water to make up 50.0 ^il

SOUTHERN TRANSFER AND HYBRIDIZATION WITH VIRAL GENE PROBE

Preparation of Southern blot

After carrying of electrophoresis, the DNA was transferred to the blotting membrane either by capillary transfer or through vacuum using the vacuum blotting apparatus (Vacu Gene^"^ XL Pharmacia).

1. Capillary transfer method

After the completion of electrophoresis. DNA in the agarose gel was denatured by placing the gel in a tray containing the denaturation solution, with slow shaking for 30 min. After denaturation, the gel was soaked in neutralization solution and agitated Materials and Methods 48

slowly for 30 min. Thereafter, the DNA was transferred onto the membrane by assembling the gel and the membrane in the following manner.

The two chambers of the electrophoresis tank were partially filled with the transfer solution (lOX SSC). A wick of Whatman 3MM paper was soaked in lOXSSC and placed over the gel platform of the chamber with its ends submerged in the solution. Three more sheets of Whatman 3 MM filter paper slightly bigger than the gel, pre-soaked in lOXSSC were placed over the wick. The gel was carefully inverted and placed over the Whatman sheets avoiding any air bubbles between the Whatman paper and the gel. Blotting membrane was cut to the gel size, wetted in water and placed over the gel careftilly to avoid bubbles. Three sheets of Whatman 3 MM paper (cut to the size of the gel and wetted in lOXSSC) were placed over the membrane. A stock of dry blotting papers (8-10 cm height) of the same size as the gel, were placed over the 3MM paper and approximately 500 g weight was placed on top of the stock. The transfer was allowed to take place for 14-16 hr.

After transfer, the blotting paper stock was removed, membrane was marked so as to indicate the orientation and rinsed in 2XSSC briefly. The membrane was then placed between the folds of the 3 MM sheet briefly (to remove buffer but not dry it) and was fixed by cross linking with UV for 7 min. The blot (wrapped in the cling film) was then stored between folds of a filter paper at room temperature till hybridization was to be performed.

Vaccuum transfer method

The apparatus was washed with distilled water and rinsed with sterile water. The blotting membrane, that was cut slightly larger than the gel, was wetted in distilled water and placed over the support screen. The gel was carefully placed over the membrane so as to avoid bubbles. It was then subjected to denaturation (15 min) and neutralization (15 min) successively by pouring the respective solution over the gel for the specified interval of time under vacuum (50-50 mbars). The transfer solution (lOXSSC) was then poured over the gel and was allowed to stay under vacuum for 45 min. After the transfer was complete, vacuum was turned off and the gel was slowly removed from over the membrane. The membrane was marked properly and rinsed in 2XSSC briefly. It was then placed between the folds of the filter paper, dried briefly Materials and Methods 49

and wrapped in cling film. DNA was cross-linked to the membrane by 7 min exposure to UV. The membrane was stored at room temperature till hybridization is to be performed.

Denaturation solution 1 M NaCl and 0.5 M NaOH. Neutralization solution 1.5 M Tris HCl (pH 8) and 3 M NaCl. SSC (20X): 3 M NaCl and 0.3 M tri-sodium citrate. Geminiviral probe preparation

Heterologous probe preparation from the cloned geminiviral DNA (ITLCV- Hallan, 1998). For heterologous probe prepration, PCR was done with geminiviruses specific coat protein primers. The PCR product was electrophoresed on 1% LMP agarose gel. The viral DNA fragment was excised from the gel, purified and then denatured by incubating in boiling water bath for 10 min for probe preparation. The reaction was setup as follows :-

Denatured DNA (~200ng) 10^1 Random Primer (75ng) 1 -2|al Now put the eppendorf in boiling water bath for 3 min, quick chill on ice, check the volume and make up to 10 or 1 1|J1 if necessary. Now add,

DTT(20mM) 3.0^1

I OX RP buffer (Random Primer buffer) 3.0 ^il

dNTPs mix (-CTP) (5.5 mM each) 2.0 |al

Klenow enzyme (5U) 1.0 \x\

a-P" dCTP (10 ^ici/^il) sp. 1.0 ^1

Water make up to 30.0 )J,1 The reaction was incubated at 37°C for three hr. To this, equal volume of "buffer A" was added. The mixture was then denatured by incubating the tube in boiling water bath for 5 min. The contents were immediately cooled on ice before adding to the hybridization bottle.

Homologous viral probe preparation as described by (Srivastava et al., 1992). Using this method "viral genome specific" radiolabelled probe can be prepared directly from the total DNA obtained from the leaves of infected plant, exploiting the ssDNA Materials and Methods 50

genome of geminiviruses. Apart from the ss form of the viral DNA, the nicked form which has discontinuity in one of its strand (and hence has single stranded region) will also be labeled by the technique.

Total DNA was isolated from the plant tissue by (Dellaporta et a/,. 1983) method. Before preparation of the probe, the nucleic acids were treated with RNase. Phenol extracted and precipitated with 2.5 volumes of absolute ethanol. 2-5 i^g of precipitated nucleic acids were taken for probe preparation without any prior denaturation. Reaction mixture was assembled as described above for heterologous probe preparation. Rest of the steps were also described for the heterologous probe preparations.

DNTPs mix (for a^^-P dCTP as the radioactive molecule from 100 mM stock of dATP, dTTP and dGTP) 1+1+1+27 ^l water. 4.5 \i\ of this mix was used for one reaction.

10 X RP buffer 500 mM HEPES (pH 6.6, adjusted with NaOH) and 100 mM MgCb.

Buffer A 500 mM Tris HCl (pH 7.5), 500 mM NaCl, 5 mM EDTA and 0.5% SDS.

Hybridization of Southern blotted DNA

Hybridization of the Southern blots with labeled probes was carried out abiding by manufactures instructions.

For Zeta probe membrane (Bio-Rad USA), the pre-hybridization / hybridization solution contained.

Formamide 50% (v/v) Na2HP04, (pH 7.2) 120 mM

NaCI 250 mM

EDTA( pH 8.0) ImM

SDS (w/v) 7%

While the Hybond (Amersham) membrane, the composition of the Pre-hybridization / hybridization solution was. Materials and Methods 51

Formamide (50% v/v) 7.50ml SSC (20x) 3.75ml Denhardts solution (5 Ox) 1.50ml SDS(w/v)(10%) 0.75ml

Water makes up to 15.0 ml The Southern blot or the Southern blotted transferred membrane was first put in a hybridization bottle keeping the side onto which DNA was transferred towards inside. Pre-hybridization solution (approximately 100 |il/sq cm of membrane) was added to the bottle. After ensuring that no air bubbles were present between the membrane and the wall of the bottle, the blot was incubated at 42°C for at least one hour in the hybridization chamber with constant rotation. The probe was boiled for 3 min in boiling water bath, chilled on ice for one minute and then added to the Pre-hybridization solution. The hybridization was allowed to occur for overnight (12-16 h) at 42°C.

Washing of the Blots

The washing of the blot was carried out as instructed by the manufactures. In brief, the following stringency washes were given to the blot to remove unhybridized probe. Stringency washes: 2X SSC, 0.1% SDS 5 min twice, IX SSC, 0.1% SDS 5 min once, O.IX SSC, 0.1% SDS 15 min once, O.IX SSC, 0.1% SDS at 42°C 15 min once (if required).

Autoradiography

After washing, the membrane was finally dried with tissue paper and wrapped in cling film. The final counts on the membrane were taken and accordingly exposed to X-ray film (Kodak) with intensifying screen (Kiran Hi-speed) in a cassette at - 70°C/RT for the required time limit. Autoradiogram was developed for 60S in Kodak developer and fixed in Kodak fixer, washed in running water and dried in air and examined.

Stripping of the probe from the blot and re-hybridization

If the blot (s) were to be used again for probing with a different probe, they were stripped off the probe in the following way as instructed by manufacturer. 0.2 M NaOH at 42°C was added to the blot and incubated at 42°C for 10 min. Repeated with Materials and Methods 52

fresh NaOH solution. Following a 15 min wash with 2X SSC, boiled solution of 0.1% SDS (w/v) was poured directly on the blots and then allowed to cool to RT. Finally the blots were rinsed twice with 2X SSC, air dried and stored between 3 MM paper. The membrane (S) from which the probe had been stripped was exposed to X-ray film (overnight) so as to check for any remaining radioactivity.

Preparation of competent cells of E.coli strain DH5a or XLI Blue

For introduction of recombinant plasmid into E. coli, competent cells were prepared as described in the procedure below.

1. A single colony of E.coli (strain DH5a or XLI blue) was picked up from a petridish plate of Luria agar (LA) containing antibiotic Ampicillin (100 mg/L cone) or Tetracycline (12.5 mg/L cone.) and used to inoculate 5 ml Luria broth (LB).

2. This was incubated at 37°C for overnight with shaking. 100 |J,1 of this over-night grown culture was used to inoculate to 100 ml LB in 500 ml flask and incubated at 37°C with vigorous shaking (300 rpm/min). 3. When the absorbance of culture at 600 nm reached 0.4, the culture was chilled on ice for 30 min, transferred to a pre-chilled SS34 tube and centrifuged at 4,500 rpm at 4°C for 5 min in a SS34 rotor of Sorvall refrigerated centrifuge. 4. The medium was discarded fully and the pelleted cells resuspended in ice cold 20ml0.1MCaCl2. 5. After keeping on ice for 30 min, the cells were pelleted again as before and resuspended in ice cold 20 ml 0.1 M MgCh. Cells were again kept on ice for 30 min and then centriftiged as before. 6. The supernatant was discarded and the cells were finally resuspended in ice cold 4 ml 0.1 M CaCl2 containing 10% glycerol (v/v). 7. The cells were stored at -70°C in aliquots of 200 [i\ each in eppendorf tube before being used for transformation.

Transformation of competent cells

Frozen competent cells were thawed on ice or 200 \i\ of freshly prepared cells were taken for transformation. Contents of the ligation reaction or (the DNA to be Materials and Methods 53

transformed) were mixed with competent cells and incubated on ice for 45 min. The tubes were then incubated in a water bath set at 42°C for exactly 90 S. The cells were immediately chilled on ice for 3-5 min. Tubes were then kept at RT and 800 ^il of LB was added to each tube. The tubes were incubated at 37°C for 1 h for the cells to recover. Cells were finally plated for the selection of transformants on LA plate containing appropriate antibiotic.

Selection of transformants carrying inserts by a-complementation

Using a sterile glass spreader, LA plates containing antibiotics Ampicillin (100 mg/L) and Tetracycline (12.5 mg/L) were coated with 40 \i\ of stock solution of X-gal (20 mg/ml) and 8 \i\ of isopropylthio-P-D-galactosidase (IPTG 200 mg/ml). The plates were allowed to dry for at least 2 h before use. 200 \A of each of the transformed cells were spread on these plates uniformly with a sterilized glass spreader. The remaining cells were briefly centrifuged for 15-20S, 600 fxl of the supernatant was discarded leaving about 200 \i\ in which the pellet was resuspended. These cells were also spread on fresh plates as above and incubated at 37°C overnight. All the white clones in the transformants of ligation reaction, were picked up and streaked on master plates of LA containing antibiotics as described above.

Characterization of the positive clone

Plasmid preparation of the positive recombinant clone was done for its further characterization.

Mini preparation method:-

1. Small scale isolation of plasmid DNA was based on the alkaline lysis method of (Bimboim and Doly 1979) as described in (Sambrook et al, 1989). 2. A single colony was inoculated in 5 ml LB medium containing appropriate antibiotic and grown overnight at 37°C with continuous shaking at 200 rpm in shaker. 3. 1.5 ml of the overnight grown culture was transferred to a micro centrifuge tube and cells were pelleted at 10,000 rpm for 45 S at 4°C.

4. The supernatant was completely discarded and cells were suspended in 100 f^l cold Sol. 1 (GTE buffer) by gentle vortexing. Materials and Methods 54

5. To the suspension, 200 \i\ of freshly prepared Sol. II (alkaline SDS) was added and the cells were lysed by gently inverting the tube several time and then kept at RT for 5 min.

6. To the lysate, 150 \i\ of ice cold Sol. Ill (potassium acetate solution: 3 M potassium and 5 M acetate) was added and the contents mixed gently as described above and kept on ice for 10 min. 7. The tubes were then centrifuged at 12, 000 rpm in a microfuge for 10 min at 4°C. The pellet (containing chromosomal DNA and salts) was discarded and the supernatant was extracted once with equal volume of phenol: chloroform: iso- amyl alcohol (25:24:l)(v/v) as described previously. 8. The DNA in the aqueous layer was precipitated by addition of 0.1 volume of 3 M sodium acetate, (pH 5.2) and two volumes ethanol precipitated by keeping it on ice for 30 min.

9. The precipitated plasmid DNA was collected by centrifuging at 12,000 rpm for 15 min at 4°C, washed with ice cold 70% ethanol and dried. The nucleic acid pellet was dissolved in 100 f^l TE and the plasmid DNA was stored at -20°C.

Sol. I (GTE) 50 mM Glucose, 10 mM EDTA, 25 mM Tris HCl, (pH 8.0).

Sol. II (Alkaline SDS) 1% SDS and 0.2 N NaOH

5 M potassium acetate 49 g potassium acetate per 100 ml solution.

Sol III (5 M potassium acetate for alkaline lysis) 5 M potassium acetate 60 ml, 11.5 ml glacial acetic acid, 28.5 ml water for 100 ml.

3 M sodium acetate 40.8 g sodium

Isolation of the DNA fragment from low melting point (LMP) agarose gel

1% LMP agarose gel in IXTAE buffer was cast, similar to that described earlier. The DNA was mixed with loading dye and loaded into the well of the LMP agarose gel. It was then subjected to electrophoresis at 60 volts for the appropriate period. All steps, starting from the casting of the gel, were performed at 4°C. The gel was then stained with ethidium bromide at 4°C for 30 min and visualized in UV light by placing it into a cling film stretched above the UV transilluminator plate. The Materials and Methods 55

agarose gel slice, with the desired band of the DN A wk^ then cut with a ^teri'Ie blade and stored at 4°C.

The tube containing the gel piece was incubated at 65°C for at least 10 min to completely melt it. Along with it, a tube containing phenol was also incubated at 65°C to heat it. Sterile TE (pH 8.0) was added to make up the volume of the mealted agarose to around 500 i^l and the tube was incubated for additional 10 min at 65°C. Equal volume of hot phenol was added and mixed properly by pipetting and again incubated at 65°C for 10 min. It was then centrifuged at full speed for 20 min at 10°C, slowly cooling the chamber of the centrifuge. The supernatant was carefully transferred into a fresh tube without disturbing the interface. The phenol extraction step was repeated twice and the supernatant was extracted thrice with 2 volumes of IXTAE saturated diethyl ether. Finally, the aqueous phase was spun under vacuum to remove the residual ether. It was then supplemented with 5 M NaCl (0.2 M final concentration) and precipitated by adding 2.5 volumes of absolute ethanol and kept at -20°C overnight.

SEQUENCING

Single clone was sequenced at both the ends (initiation and termination) by Sanger dideoxy chain termination method (Sanger et al., 1977). The actual reaction was carried out using the T7 sequencing kit^^ (Pharmacia Biotech). Primers for sequencing were obtained either from Pharmacia (Reverse, Universal, T7 and SP6 primers) or Stratagene (KS and SK primers). Electrophoresis was carried out in Sequi-Gen Nucleic acid sequencing cell (Bio-Rad) with 17x15 cm actual gel area.

Preparation of the sequencing gel

Glass plate, spacers, comb and clamps were thoroughly washed, rinsed with deionized water and then with ethanol. The IPC (Plate fixed to upper buffer chamber) was coated with 5% (v/v) dimethyldichlorosi- lane (repel silane) solution in chloroform or hexane. The spacers (0.4 mm or 0.25 mm thick) were placed between the two plates and the plates were then held firm with two clamps. Gel solution was prepared by mixing the two solutions, urea acrylamide and urea-TBE. The solution containing acrylamide was deionized by stirring with Dowex MR-12 (2 g for 200 ml solution) for 1 hr and then filtered. The two components were mixed appropriately to prepare a Materials and Methods 56

solution having the desired acrylamide concentration. For example for an 8% gel the two were mixed as follows.

Urea acrylamide 28 ml Urea TBE 42 ml Total 70 ml To 20 ml of the above solution 100 |il of 25% ammonium presulfate (APS, prepared freshly) and 100 |al TEMED were added, mixed and immediately poured in the gel sealing strand. Assembled gel plates were kept ready and were immediately fixed into the strand to seal the gel apparatus. It was allowed to strand for 5-10 min so as to seal the gel apparatus properly. To the remaining 50 ml of the above prepared gel solution 100 1^1 25% APS and 50 |j,l TEMED was added. The solution was poured slowly into the gel apparatus (keeping the solution on ice so that it does not polymerize fast) and taking care not to introduce any air bubbles in the gel. Comb of appropriate thickness was introduced and the apparatus was held at 45° during pouring as well as after that till polymerization was complete, the apparatus was detached from the sealing tray and fixed into the lower buffer chamber. The chamber attached with the IPC plate as well as the lower chamber was tilled with IX TBE buffer. The gel was pre-run for at least 1 hr at 50 W constant power so that the gel attained a temperature of ~50°C (the optimal temp, at which sequencing is done).

Urea Acrylamide (200 ml) 38.6 g acrylamide, 1.34 g bis-acrylamide, 84 g urea and

20 mil OX TBE.

Urea TBE (200 ml) 84 g urea and 20 ml 1 OX TBE.

Sample preparation

3-4 i^g double stranded (ds) or 1.5-2.0 j^g single stranded (ss) DNA purified by the use of helper phages was taken for sequencing of the clones and sub clones, ss and ds DNA were processed differently initially as there is no need to denature the ssDNA while dsDNA needs to be denatured before aimealing step. After armealing step both were treated in a similar manner. Materials and Methods 5 7

Denaturation of double stranded DNA template

Concentration of the dsDNA template was adjusted so that 32 |^1 contained 3-4 fig of it. To this 8 |il of 2 M NaOH was added, mixed properly and incubated at 25''C for 10 min. To this, 4 \i\ water and 7 )j.l of 3 M sodium acetate (pH 4.8) was added followed by the addition of 120 f^l ice-cold absolute ethanol. This was incubated at - 20°C for 1 hr. Precipitated DNA was recovered by centrifugation at 12,500 rpm in a microfuge for 20 min. The DNA pellet was washed with 80% alcohol, dried and resuspend in 10 |al sterile water.

Annealing of the primers

To the denatured ds DNA template (10 ^1), 2 \i.\ annealing buffer and 2 |xl primers (-50 ng) were added. The mixture was incubated at 65°C for 5 min. In case of the ssDNA, it was mixed with -10 ng primers, 2 j^l annealing buffer and incubated at 60°C for 5 min. Thereafter, incubation in both the cases was carried out at 37°C for 10 min followed by a further 10 min incubation at 25°C. After the annealing was complete both were taken for the next step.

Labeling Reaction

To the tube containing annealed template and primers (14 )al), following component were added.

Labeling mix for dCTP 3)J.1 Labeling dNTPs (a"-P-dCTP or a^' S-d CTP) 1 ^1 (10 |i ci) Diluted T7 DNA polymerase (1 |xl T7 DNA polymerase) 2 ^il (Diluted with 4 \i\ enzyme dilution buffer) This was incubated at 25°C for 5 min. Materials and Methods 5 8

Termination reaction

While the labeUng reaction was in progress. 2.5 ^il of short sequencing mixes ('A' mix short, 'T' mix short, 'G' mix short and 'C mix short) were dispensed into four tubes labeled as A.T.G. and C. respectively. 4.5 )x\ of labeling reacting mix (after 5 min incubation) was distributed into each tube which were pre-warmed at 37°C for 1 min. The component were mixed and the tubes were incubated at 37°C for 5 min. After the reaction was complete, 5 |il of stop solution was added to each tube.

Electrophoresis

After pre-run, the sample comb was removed and the wells were washed thoroughly (with buffer) with the help of a syringe. The reaction mixture (12 ^1) was divided into two tubes for 1^' (7 i^l) and 2"** (5 |j.l) loading. Samples for first loading were denatured by incubation at 90-95°C for 3 min and immediately loaded into the wells. Electrophoresis was carried out at constant temperature of 50°C (achieved with the help of a temperature probe). After 2-3 hr (or when the bromophenol blue reached the bottom of the gel samples)for second loading were denatured and loaded into respective wells. Electrophoresis was continued further for an appropriate amount of time (decided by the number of base pairs between the cloning site and the primer annealing position).

Autoradiography of the gel

After the electrophoresis was over, electrodes were disconnected, gel apparatus was removed from the lower gel tank and kept in a horizontal position. Clamps were removed and the glass plates separated taking care not to detach the gel from the out plate. For a P gel, the gel was directly removed fi-om the outer plate by placing 3 MM sheet over the moistened gel surface. The gel was wrapped in cling film and exposed to X-ray film in a cassette at -70°C or -20°C for 8-12 hrs. For a^^S the gel along with outer plate was fixed by placing in a tray of fixing solution (containing sufficient fixing solution so as to submerge the gel). It was allowed to stay in the fixing solution for 20 min. The fixing solution was drained properly and the gel was then transferred to Whatman 3 MM sheet, wrapped in cling film, dried in a vaccum gel drier (LKB) for 1- 1:30 . After it had dried completely. Cling film was removed from the gel and the 3 Materials and Methods 59

MM sheet containing the gel was exposed to the X-ray film for 12-24 hr at -70°C. Signals on the film was developed and fixed.

Computer analysis of the sequencing data

Sequencing data obtained was manually fed into the computer. SEQIN program of PC/GENE software was used for this purpose. Other programme of the software, i.e., TRANSL, NALIGN, PALIGN, CLUSTAL, RESTRI, NAMANIP etc. were used for fiirther analysis of the sequence data. Results RESULTS

BIOLOGICAL CHARACTERIZATION

Natural symptoms

Naturally infected plant of Calendula officinalis L. growing in the experimental field of Aligarh Muslim University and many gardens in Aligarh showed well marked symptoms of yellowing of the vein during 2002-2003. The incidence of disease was less (about 10-15%). The symptoms of the disease were yellowing of the veins, shortening of the leaf and petioles, thickening/ swelling of the vein and stunting of the whole plant (Fig 4.1). Severely infected plant of calendula also showed mosaic symptoms on the calyx, corolla and reduction of the florets and flower buds (Fig 4.2). It was also noticed that the infected plants bear less floral buds in comparison to healthy plants.

TRANSMISSION

Mechanical transmission

Preliminary transmission experiment was carried out using naturally infected Calendula officinalis L. as donor and seedlings of Calendula offiicinalis, Lycopersicon esculentum, Datura stramonium, Nicotiana tabacum cv. Samsun NN and Nicotiana tabacum cv. White Burley as recipient hosts. Inoculation of sap from infected C. officinalis prepared in 0.1 M potassium phosphate buffer (pH 7.0) revealed that the pathogen was not sap transmissible as no local or systemic symptoms appeared on any of these plants.

Aphid transmission

The non-viruliferous aphids (Aphis gossypii and Myzus persicae) were tried as vectors for transmission of the calendula virus in non-persistent manner to different host plants mentioned earlier. However, these vectors were not able to transmit the calendula virus to any of the test plants.

Whitefly transmission

Non viruliferous whiteflies (Bemisia tabaci) were given an acquisition access period for 24 hr to a twig of naturally infected calendula plant in an insect proof cage. T3 C CO $. s c o CO E o a E (0 o CO (0 o E c I w CM OT ^ O

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After 24 hrs these viruliferous whiteflies were removed from the infected twig, and were allowed to feed on healthy calendula seedlings (at two-four leaf stage) for 48 hr on an average of five whiteflies to each seedlings. After the completion of inoculation access periods, the surviving whiteflies were killed by spraying with 0.1%(v/v) insecticide (Dimecron) and the seedlings were kept in an insect proof cage. Whiteflies inoculated plants were observed periodically for the appearance of disease symptoms. The first symptom started appearing 25 days post inoculation and the inoculated seedlings of calendula showed yellow vein symptoms like the naturally infected plants.(Fig 4.3). The above biological characterization indicates that the virus isolate infecting calendula in nature, may be of whitefly transmitted virus.

Host range studies

Calendula yellow vein disease was successfully transmitted by whiteflies to Lycopersicon esculentum. Capsicum annuum, Nicotiana tabacum cv. White Burley, but not to Datura stramonium. The seedlings of tomato, chilli and tobacco showed leaf curling, shortening of intemodes and petioles and stunting of the entire plants after 35 days of post inoculation (Fig 4.4). However, in case of datura, no symptoms were produced and the plant remained healthy even after 45 days of post inoculation (Table 4.1). The inoculated plants were further checked by PCR amplification with begomovirus specific TLCV coat protein primers (Hallan, 1998). The PCR products were electrophoresed on 1% agarose gel and PCR results gave positive amplification (~750bp) in tomato, chilli and tobacco but no such band could be obtained in datura plant, indicating that datura is a non-host of calendula virus (Fig. 4.5a).

To confirm the specificity of PCR amplicon, the DNA from the same agarose gel was transferred to Hybond nylon membrane and hybridized with well-characterized radiolabelled begomovirus specific probe (ITLCV, Hallan, 1998). The probe hybridized well with blotted DNA under high stringency condition and developed strong signals on X-ray film (Fig 4.5b).

Based on PCR with begomovirus specific TLCV coat protein primers (Hallan, 1998), and Southern hybridization, virus isolate infecting calendula in nature seems to be a begomovirus of family Geminiviridae. u

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Whitefly inoculated Symptoms appeared Symptoms appeared Time plants in No. of plants tested (dpi) Calendula officinalis Yellowing of the vein, 3/5 25 stunted growth of plant. Lycopersicon esculentum Curling of the leaf, 2/5 35 stunted growth of plant. Capsicum annum Curling of the leaf, 2/5 35 shortening of internodes and petioles. Nicotiana tabacum cv. Curling of the leaf, 3/5 35 White Burley shortening of internodes and petioles. Datura stramonium No symptoms appeared 0/5 45 = £ c o

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Seed transmission

In seed transmission test, the seedlings grown from the seeds collected from naturally infected calendula plants in insect proof condition, did not show any type of symptoms up to their flowering stage. Furthermore, total DNA isolated from these plants and subjected to PCR amplification with begomovirus specific TLCV coat protein primers (Hallan, 1998), also gave no positive amplification, revealing that the calendula virus is not seed transmissible.

Experiments done in molecular virology lab, N.B.R.I. Lucknow.

MOLECULAR CHARACTERIZATION

DNA-Isolation

Total DNA was isolated from newly emerging naturally infected tissues of calendula by following the method described by Dellaporta et al., 1983, which yielded a good quality and quantity of DNA prepration as checked by taking its best CD. at 260/280 which is 1.8 and concentration was also checked by the 1% agarose gel electrophoresis.

PCR with Begomovirus coat protein (TLCV) specific primers

PCR amplification was performed with TLCV coat protein gene begomovirus specific primers (Hallan, 1998), using total viral DNA isolated from naturally infected calendula plant.. Results of electrophoresis, on 1% agarose gel (prepared in IX TAB buffer) showed the expected size amplicon of -750 bp in infected calendula however, no such band was observed in healthy sample (Fig 4.6a).

To confirm the authenticity of the PCR product as begomoviral origin, the DNA from the same agarose gel was transferred to Hybond membrane (Amersham - Pharmacia, U.K.) for hybridization with a-P labelled probe from a clone of (CP gene) of a well characterized begomovirus (ITLCV; Hallan 1998). The probe hybridized well with blotted DNA under high stringency conditions, developed strong signal of hybridization on X- ray film. Virus infecting calendula in nature seems to be a begomovirus,(Fig 4.6b). •o c (8 MM Q: o Ul £ <-> •| B CNJ T3iS o o m li w w <- SI flJ :u>-^= 3 T3 -o 0) C

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PCR with P-DNA detecting universal primers

The PCR product obtained with (3-DNA specific primers (BetalF and BetalR- universal primers) electrophoresed in (IXTAE) 1% agarose gel showed approximately -650 bp bands (Fig 4.7a) (about half of the P-DNA genome) suggesting the association of a P-DNA satellite molecule of low molecular weight to that of the 1.3 Kbp. Such low molecular weight P-DNA satellite molecules have also been found associated with another begomovirus i.e. Tomato leaf curl virus (ToLCV) earlier reported by Dry et al, 1997.

Furthermore, the specificity of the PCR product as P-DNA was also confirmed by Southern hybridization of the blotted DNA from the same agarose gel. The homologous probe hybridized well with blotted DNA membrane under high stringency condition, developed strong signal of hybridization on X-ray film (Fig 4.7b), confirmed that PCR amplicon was of P-DNA origin suggesting the association of P-DNA with calendula isolate.

Cloning of PCR product in pGM-T-Easy Vector and selection of clones

The ~750bp PCR product obtained with begomovirus specific TLCV-CP primers, (Hallan, 1998) was LMP eluted and used for the ligation into a suitable cloning vector pGMT Easy vector system-1, Promega Corporation, USA, (Fig. 3.1 and 3.2). The cloning kit vector and PCR product used for the ligation reaction in 3:1 molar ratio incubated at 4°C over night. Over night ligation reaction was done as described in M&M. The ligation mix was used to transform of competent E. coli DH5a cells. In order to select clones containing the recombinant plasmid, X-gal/IPTG was spread on the antibiotic (Ampicillin lOOmg/L) supplemented plates, which allow the blue/white color selection of recombinant clones. Only the white colonies were selected for further analysis.

Screening of transformants by PCR

To identify that the clones containing insert of coat protein gene, only white colonies were selected from the selection plates and serial number was assigned to them as 1 to 30. These 30 white clones were inoculated in 5 ml LB containing Ampicillin and incubated at 37''C and 200-rpm over night. The plasmid DNA isolated •D 0) CO.

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from these colonies were prepared as detailed in M&M and pellet was suspended in 50 ^il sterile water.

To check the presence or absence of coat protein gene in these white clones, the plasmid DNA isolated from these white clones were screened by PCR with the begomovirus TLCV coat protein specific primers (Hallan, 1998). The PCR amplification reaction resulted in positive amplification (-750 bp) in 11 out of 30 clones as positive recombinants (Fig 4.8a).

Furthermore, to confirm the specificity of PCR amplified recombinants clones as insert of coat protein gene, the electrophoresed gel was transferred to Hybond membrane and hybridized with well characterized begomoviral probe (ITLCV, Hallan, 1998). All the 11 clones screened as positive recombinants by PCR also developed the strong signals on X-ray film. (Fig 4.8 b).

Screening of clones by restriction digestion

Few clones were digested with the EcoRX restriction enzyme (sides ^t both the ends in the vector) for over night at 37 °C in water bath. The restricted DNA was electrophoresed on 1% agarose gel, which showed presence of two bands of ~3.0kb (vector) and ~0.75kb (insert) (Fig 4.9a). Furthermore, to check the authenticity of the restricted bands, 1% electrophoresed gel was Southern transferred to the nylon membrane and hybridized with the radiolabelled begomoviral probe (ITLCV, Hallan, 1998). Strong signals were developed on X-ray film under high stringency condition.(Fig 4.9b). Therefore, based on PCR, Southern blot hybridization and restriction digestion of positive clones, it was proved that the clones from calendula isolate were positive and right clones which may be processed for sequencing. o E N>« C o c o '« u 'SM m O k

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Sequencing of positive recombinant clone

The positive recombinant clones were confirmed by PCR followed by Southern hybridization and their restriction digestion. One such positive clone was sequenced using universal primers (T7 & SP6) from both the initiation and termination ends, (procedure is describe in Material & Methods). The nucleotide sequence data was corrected by autoradiography.

Sequence analysis

The nucleotide sequence data indicated that CP gene of calendula isolate encompassed the complete Open Rading Fram (ORF) of 519 bp including the initiation codon (ATG) and termination codon (TAA) (Fig 4.10). The deduced amino acid sequence of coat protein consisted of 173 amino acids (Fig 4.11). The nucleotides sequence of coat protein obtained was deposited in gene bank under the accession number AY887174.

Amino acid alignment

Clustal multiple sequence alignment of amino acid of calendula isolate revealed the presence of 13 unique sites out of 253 deduced amino acids. These positions were identified as "A2S" ("A" in place of "S" at 2 position), "K180R" ("K" in place of "R" at 180 position), "R181K" ("R" in place of "K" at 181 position), and "LKIILR- MRW"218 to 227"YENHTENALM" ("LKIILR-MRW" in place of YENHTENALM" at 218 to 227 position (Fig 4.12).

However, there were no unique sites from 61-120 and 241-253 amino acid. They were found to be identical, indicating as conserved region in the CP of calendula isolate.

Percent identities of nucleotides (nt) and deduced amino acid (aa) sequence between Calendula isolate and related viruses from all over the world

Results of pairwise nucleotide blast search alignment of calendula isolate with the sequence data available in the Gene bank revealed a maximum of 96% (737/769) sequence similarities with Tobacco curly shoot virus (ToCSV) (AJ420318) and minimum 83% (638/745) with Euphorbia leaf curl virus (EuLCV) (AJ558121). The sequence similarities were 95% (730/769) with Ageratum enation virus (AgEV) Fig. 4.10. Complete nucleotide sequence of coat protein gene of Calendula isolate.

1 ATGTACAGGATGTACAGAAGTCCAGATGTCCCTAGAGGATGTGAAGGCCC 51 ATGTAAGGTCCAGTCGTTTGAGTCCAGACATGACATTCAGCATATAGGTA 101 AAGTCATGTGTGTCAGTGATGTTACGCGTGGAACTGGGCTGACTCACCGA 151 GTGGGTAAAAGGTTTTGTGTTAAATCCGTTTATGTCTTGGGTAAGATATG 201 GATGGA CGAGAATATTAAGACCAAAA ATCACACGAA CAGTGTGATGTTT 251 Mil lAGTTAGGGATCGTAGACCTGTGGATAAACCTCAAGATTTTGGTGAG 301 GTGTTTAACATGTTTGATAATGAGCCCAGTACGGCTACTGTGAAAAATGT 351 TCATCGTGATAGGTATCAAGTACTTCGGAAATGGCATGCAACTGTGACCG 401 GTGGACAATATGCGTCAAAGGAACAAGCTCTTGTGA AGAAGTTTGTTAGG 451 GTTAATAATTATGTTGTGTATAACCAGCAAGAAGCTGGCAAGTTGAAAAT 501 CATTCTGAGAATGCGTTAA

(Total nucleotide = 519 base pairs) Base Count: A-156, T-149, C-76, G-138 The initiation (ATG) and termination (TAA) codon are written with different colour.

1 MYRMYRSPDVPRGCEGPCKVQSFESRHDIQHIGKVMCVSDVTRGTGLTHR 51 VGKRFCVKSVYVLGKIWMDENIKTKNHTNSVMFFLVRDRRPVDKPQDFGE 101 VFNMFDNEPSTATVKNVHRDRYQVLRKWHATVTGGQYASKEQALVKKFVR 151 VNNYNAA'NQQEAGKLKIILRMR*

Total Amino acid residues= 173

Fig 4.11 The deduced amino acid sequence of coat protein gene of Calendula isolate. 1 60 CoLCV S ToLCKV S ToGV s- CYW V- AgBemoV-Pak R loCSV S ToCSV-Y S- AgEV S- TLCV S- PaLCV S PLCV S- EuLCV S- ICaMV S- ToLCV 180 CoLCV R "loLCKV R ToGV CYW K AgBemoV-Pak R ToCSV R ToCSV-Y R AgEV R TLCV R PaLCV R PLCV R EuLCV R IcaMV R ToLCV -R^ 81 240 CoLCV K YENHTENALM- ToLCKV K YENHTENALM- ToGV CYW fR+ LKIILR-MRW—- AgBemoV-PakK YENHSENALM ToCSV K YENHSENALM ToCSV-Y K YENHSENALM- AgEV K- YENHSENALM- luCV K- YENHSENALM. PaLCV K YENHSENALM PLCV K YENHTENALM. EuLCV K YENHTENALM- ICaMV K+ YENHTENALM4 ToLCV YFNH.SFNAI M Fig 4.12 Amino acid sequence alignment of OP gene of Calendula isolate related with other Begomoviruses OP gene. Dash line indicates identical sequences. Sequence unique to other Begomoviruses are indicated in box. Results 66

(AJ437618), 94% (719/769) with Papaya leaf curl virus (PaLCV) (AJ436992), 93% (720/769) with Tomato leaf curl Bangladesh virus (TLCV) (AFl 88481), 92% (703/752) with Ageratum begomovirus Pakistan (AgBegV-Pak) (AJ810825), 91% (707/769) with Cotton leaf curl Kokham (CoLCV) (AJ002449) and 87% (684/769) with Pepper leaf curl Bangladesh virus (PLCV) (AF314531). 96% sequence similarities of the virus isolate was also observed with Tomato geminivirus-China (TomGV-china) (AJ566745) but only in a small stretch (212/216) which may be insignificant.

Since the present virus, possess maximum similarities with above viruses, which belong to the geminivirus group, our virus seems to be a geminivirus.

Pairwise nucleotide alignment of the virus isolate from calendula with the selected geminiviruses revealed maximum similarities 96% with ToCSV and TomGV- China virus and minimum 77% with TLCV-Banglore. However, similarities in descending order are 95% with AgEV and ToCSV-Y, 94% with PaLCV, 93% with TLCV, 92%) with AgBegovirus-Pakistan and 91% with CoLCV. Best similarities were less than 90%.(Table 4.2). ''

Pairwise amino acid alignment of the virus isolate from Calendula revealed maximum 97% with TomGV-China, 95% with ToCSV-Y, 94% with AgEV, ToCSV, TLCV and 93% with PaLCV, PLCV (Table 4.3).

On the basis of pairwise alignment of nucleotide as well as amino acid, present virus isolate (calendula) showed maximum similarities 96-97% with ToCSV and ToGV-China, it may be concluded that the virus isolate from calendula is similar to that of ToGV-China and ToCSV geminivirus.

Phylogenetic analysis

The nucleotides and amino acids sequences were aligned to generate dendrogram. The nucleotide dendrogram showed, the virus isolate of calendula closest with ToCSV. The TomGV-China virus was found to be in the same cluster but it was less closer than that of ToCSV-Y (Fig 4.13). 00 N« ^»J si «s u^^u

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CLCV

ToLCKV

ICaMV

PLCV

EuLCV

CoLCV

PaLCV

TLCV

TomGV-china

ToCSV-Y

AgEV

AgBegemoV-Pak

CYW

ToCSV

Fig 4.13 Phylogenetic relationship of Calendula isolates with other reported Begomoviruses, based on the alignment of the nucleotide sequence of the coat protein gene created by PHYLIP 3.5 programe Results 67

The amino acid (aa) dendrogram of the virus isolated from calendula showed close association with ToGV-China, however other virus ToCSV-Y was found in the same cluster but less closer than ToGV-China. (Fig 4.14).

These results of dendrogram of nucleotide and amino acid showed close similarities of the virus isolate of calendula with ToGV-China and ToCSV, both happens to be the begomovirus, therefore present virus was identified as a begomovirus similar to the ToGV-China virus.

On the basis of results obtained by virus transmission, PCR amplification, Southern hybridization, sequence alignment of nucleotide and amino acid and cluster analysis of CP gene of calendula virus isolate, the virus was identified as Calendula yellow vein virus (CYVV) which is similar to ToGV-China and ToCSV viruses. AgEV

TLCV

PaLCV

PLCV

CoLCV

ToLCKV

EuLCV

ICaMV

ToLCV

ToCSV-Y

ToCSV

AgBemoV-Pa

ToGV

CYW

Fig 4.14 Phylogenetic relationship of Calendula isolate with other reported Begomoviruses, based on the alignment of the amino acid sequence of the coat protein gene created by PHYLIP 3,5 programe. Discussion DISCUSSION

Calendula officinalis L. belongs to family Compositae / Asteraceae which occupies the highest position among the angiosperms or at least among the dicots in the course of evolution. There are 25 species of calendula but only two species viz. C. officinalis and C. arvense L. are found in India (Wealth of India).

Calendula is also known as pot marigold. It has great economic importance. Calendula extract has been used for antiseptic cream named as borocalendula in India.

Like other pathogens that affect the aesthetic value of ornamental plants, viruses are of significant importance due to absence of therapeutic control measure against them in plants. Viruses have economic importance due to severe losses, reduction in growth, number and size of flowers caused by them.

Economic losses due to geminivirus infection in cotton (Briddon & Markham; 2000), tomato (Moffat; 1999), cassava (Thresh; 1998) and grain legumes (Verma; 1992) have been observed in India as well as abroad. More than 80% of the known geminiviruses are transmitted by whiteflies and belong to the genus Begomovirus, most have bipartite genome designated as DNA-A and DNA-B and infect dicotyledenous plants.

Viruses reported on calendula are Potato virus Y (Vela, 1972); Turnip mosaic virus (Lisa et al,. 1979); Calendula rosette disease (Gupta and Verma, 1983^; Tobacco mosaic virus (Vela, 1972, Hristova et al,. 1994), Cucumber mosaic virus (Lisa et al, 1979, Naqvi and Mehmood, 1980, Naqvi et al,. 1985 and Naqvi et al,. 1989) and Tomato chlorosis Cirinivirus (ToCV) (Gail et al, 1999).

Recently, a new viral disease was observed on calendula at Aligarh Muslim University, Aligarh campus and in the surrounding area. The symptoms were yellowing of the vein, shortening of the leaf and petioles, reduction in number of flowers. The disease was transmitted by whiteflies, however, the symptoms of the disease were quite dissimilar to that of a whitefly transmitted disease earlier reported by Gupta and Verma, 1983. Discussion 69

Biological characterization

The causal pathogen of calendula was successfully transmitted from naturally infected calendula to the newly emerging seedlings of calendula through whiteflies, which produced the symptoms alike to naturally infected plants.

During host range studies using whitefly vector, calendula virus was also transmitted to Lycopersicon esculentum, Capsicum annuum, Nicotiana tabaccum cv. White Burley, which produced leaf curling, shortening of the intemodes and petioles and shortening of the entire plant. Datura stramonium did not show any type of symptoms.

Based on symptomatology, host range studies and whitefly transmission, virus causing yellow vein disease on calendula was suspected to be a geminivirus. Moreover whitefly transmitted virus causing rosette disease on calendula has been reported (Gupta and Verma, 1983) but the present isolate did not produce any rosetting symptoms on calendula. Therefore it seems to be a different virus, may be a geminivirus.

Molecular characterization of calendula virus

The causal pathogen of Calendula yellow vein disease was checked by the PCR amplification with begomovirus specific TLCV coat protein primers (Hallan, 1998). The PCR resulted positive amplification of -750 bp in the plants showing symptoms of the disease. The authenticity of PCR amplification was checked by Southern hybridization using radiolabelled probe of a well-characterized begomovirus (ITLCV, Hallan, 1998). This suggests that the PCR amplification was geminiviral in nature. -750 bp PCR product obtained by TLCV-CP primers (Hallan, 1998), was cloned, sequenced and submitted in the gene bank under the accession number (AY887174).

The sequence data of clone of calendula isolate revealed that the gene was 519 bp in length and it encompassed the complete ORF with initiation codon (ATG) to termination codon (TAA). This encodes 173 amino acid residues.

Amino acid sequence alignment of calendula isolate revealed that four amino acid positions "A2S", "K180R", "R181K" and "LKIILR-MRW218 to 227 Discussion 70

YENHTENALM" were unique in the total amino acid sequence data. However, there are no unique sites from 61-120 and 241-253 amino acid.

Pairwise nucleotide alignment of the virus isolate from calendula with the selected geminiviruses revealed maximum similarities 96% with Tobacco curly shoot virus (ToCSV) and Tomato Geminivir us-China (TomGV-China), minimum similarities 77% with TLCV-Banglore. While pairwise amino acid alignment of the virus isolate revealed maximum 97% similarities with TomGV-China. Based on pairwise alignment of nucleotide and amino acid showed maximum similarities 96-97% with ToCSV and TomGV-China. Present virus isolated from calendula seems to be similar to that of TomGV-China and ToCSV geminivirus.

The dendrogram of nucleotides and amino acid showed close similarities of the virus isolate from calendula with ToCSV and TomGV-China.

The association of p-DNA molecule, which is one of the characteristics of several begomoviruses, was also checked by PCR using p-DNA specific universal primers. Results of PCR product showed -650 bp amplification which was half of the P-DNA molecule (1.3kbp). Such low molecular weight P-DNA satellite molecules have also been reported by Dry et al., 1997. Southern hybridization of PCR product with specific probes suggested the association of P-DNA with the virus isolated from calendula.

On the basis of results obtained by virus transmission PCR amplification, Southern hybridization, sequence alignment of nucleotides, amino acids and cluster analysis of CP gene, the virus has been identified as Calendula yellow vein virus (CYVV), which shows close similarity with Tobacco curly shoot virus (ToCSV) and Tomato geminivirus (TomGV-China) reported from China. A P-DNA molecule has also been found to be associated with the virus isolate. On the basis of above studies, the virus isolated from calendula comes under the group of begomovirus, genus geminivirus and family geminiviridae. *

Yellow net disease of calendula associated with Cucumber mosaic virus has been reported previously (Naqvi & Samad, 1985), however our is the first report on natural infection of calendula by a begomovirus and association of the P-DNA with virus isolate. ^ Discussion 71

Moreover, the finding on molecular characterization of the virus causing yellow vein disease on Calendula will provide data to the researchers on the genome organization of a new begomovirus. Association of a P-DNA molecule with the virus isolate may have a definite role in symptom severity or early development of symptoms as in case of Bhendi yellow mosaic disease (Usha et al,. 2003), Tomato leaf curl virus (Dry et al, 1997), Okra leaf curl virus (Mansoor et a/,.2001), Cotton leaf curl virus (Briddon et a/.,2001), Ageratumyellow vein virus (Saunders et al,. 2000).

The sequence data generated on CP gene of the virus may be utilized in developing CP mediated resistances against virus isolate in calendula as well as other economically important plants. The clones generated during the studies may be used to develop molecular diagnostic probes for sensitive and reliable detection of geminivirus in calendula, so that the healthy virus free environment may be made available to the pharmaceutical industries. Summary SUMMARY

Calendula officinalis L. belongs to family Compositae/ Asteraceae. It has great economic importance. Reports existing in the literature revealed that calendula plant is attacked by many plant viruses.

A new viral disease was observed on calendula at Aligarh Muslim University, Aligarh campus and in the surrounding areas with the symptoms, yellowing of the vein, stunted growth of plant, shortening of the leaf and petioles and reduction in the number of the flowers. However, there was no report at molecular level for the characterization of virus isolate from calendula, therefore to provide clean and healthy environment of calendula to the several pharmaceutical industries, it was essential to identify the causal pathogen and to develop the diagnostics at molecular level.

The initial biological studies revealed that the causal pathogen of calendula was successfully transmitted from naturally infected calendula to the newly emerging seedling of calendula through whitefly, which produced the symptoms alike to naturally infected plant.

The calendula isolate was tested for host range studies. The yellow vein disease of calendula was successfully transmitted to Nicotiana tabacum var. White Burley, Lycopersicon esculentum and Capsicum annuum, producing leaf curling, shortening of intemodes and petioles. While Datura stramonium did not show any type of symptoms.

Based on symptomatology, host range studies and whitefly transmission, virus causing yellow vein disease on calendula was suspected to be geminivirus.

The causal pathogen of Calendula yellow vein disease was checked by PCR amplification with begomovirus specific TLCV primers. The PCR results gave positive amplification at -750 bp in the plant showing symptoms of the disease. The specificity of the PCR amplicon was checked by Southern hybridization using radiolabelled probe of a well characterized begomovirus (ITLCV), which proved that the PCR amplification was geminiviral in nature. Summary 73

The DNA band was LMP eluted and cloned in a suitable cloning vector (pGMT Easy vector).

The sequence data of clone of calendula isolate revealed that the gene was 519 bp in length and it encompassed the complete ORF with initation (ATG) to termination (TAA) which encodes 173 amino acid residues. The nucleotides sequence was deposited in the gene bank under the accession number A Y887174.

Amino acid alignment of calendula isolate revealed that four amino acid positions "A2S", "K180R", "R181K" and "LKIILR-MRW2I8 to 227 YENHTENALM" were unique in the total amino acid sequence data. However, there are no unique site from 61-120 and 241-253 amino acid.

Based on pairwise alignment of nucleotides and amino acids i.e. maximum similarities 96-97% with ToCSV and TomGV-China. Present virus isolated from calendula seems to be similar to that of TomGV-China and ToCSV geminivirus.

The dendrogram of nucleotides and amino acids showed close similarities of the virus isolated from calendula with ToCSV and TomGV-China.

On the basis of results obtained by virus transmission, PCR amplification, Southern hybridization, sequence alignment of nucleotide, amino acid and cluster analysis of CP gene, the virus has been identified as Calendula yellow vein virus (CYVV), which shows close similarities with Tobacco curly shoot virus (ToCSV) and Tomato Geminivirus-Ch'ma. It comes under subgroup begomovirus, genus geminivirus. p-DNA molecule has also been found associated with the virus isolated from clanedula.

Molecular characterization is expected to lead a better understanding of the taxonomy of the virus. This will helps in working out how the isolate under study related to the other reported viruses from other parts of the world. References REFERENCES

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NEW DISEASE REPORT

First report of a begomovirus infecting Calendula in India

A. A. Khan^ Q. A. NaqvP, M. S. Khan^ R. Singh" and S. K. Raj:b' *

'Plant Virology Laboratory, Department of Botany, Aligart) Muslim University, Aligarh 202 002, and ^Plant Molecular Virology, National Botanical Research Institute, Lucknow 226 001, India

Calendula officinalis (family Asteraceae) is grown as an the virus infecting C. officinalis with Tobacco curly shoot annual ornamental plant in India. Yellow vein net disease vtrus (AF240675), Ageratum enation virus (AJ437618) and of Calendula wa^ observcu uu bcvcidl planlb growing in Tornalo kafturl Bangladesh virus (AF188481), respectively. gardens at Aligarh and Lucknow. Symptoms of the disease The virus isolate under study was identified as a consist of vein yellowing, shortening of leaves and peti­ begomovirus on the basis of whitefly transmission; oles, and stunting of plants. The disease was experimen­ amplification of a DNA band of the expected size with tally transmitted from naturally infeaed Calendula to b^omovirus-detecting primers by PCR; positive hybridi­ healthy seedlings by whiteflies (Bemtsia tabact), but not by zation of PCR products with a known begomovirus- mecliariical oi apiiid tiansmissioii. specific probe? and its high sequciice identity to known Total DNA was extracted from leaves of plants with begomoviruses. and without symptoms, and PCR was performed using Yellow vein net disease of Calendula associated with a pair of primers designed to the coat protein region Cucumber mosaic virus has been reported previously (Naqvi of a well characterized begomovirus. Tomato leaf curl & Samad, 1985). However, this report is the first record New Delht virus (ToLCNDV; Hallan, 1998). Agarose of the natural infection of Calendula by a begomovirus. gel electrophoresis of PCR products showed amplifica­ tion of a product of the expected size (= 800 bp) from disease-affected but not from symptomless samples. The References authenticity of the PCR amplicon was further confirmed Hallan V, 1998. Genomic organization of geminivirus causing by cross-hybndization with a ToLCNDV DNA-A probe. leaf curl in tomato {Lycopersicon esculentum). Lucknow, The PCR produa wjs cloned and partially sequenced India: Umversity of Lucknow. PhD thesis. (accession no. AY887174). Sequence analysis revealed the Naqvi QA, Samad A, 1985. Purification and properties of highest nucleotide sequence identities (95,94 and 93%) of Calendula yellow na virus. Indian Journal of Virology 1,143-6.

*E-mail: skra)[email protected] Accepted 2S February ZOOS at wunv.bspp.or$.ukJndr where figures relating to this paper can be mewed

©2005BSPP 569 Sequence of Calendula isolate submitted to Genbank:

LOCUS AY887174 770 bp DNA linear VRLOl-FEB-2005 DEFINITION Calendulla yellow net begomovirus coat protein gene, partial cds. ACCESSION AY887174 VERSION AY887174 KEYWORDS . SOURCE Calendulla yellow net begomovirus ORGANISM Calendulla yellow net begomovirus Viruses; ssDNA viruses; Geminiviridae; Begomovirus. REFERENCE 1 (bases 1 to 770) AUTHORS Khan,A.A., Khan.M.S., Singh.R. and Raj,S.K. TITLE Calendulla yellow net begomovirus isolated from India JOURNAL Unpublished REFERENCE 2 (bases 1 to 770) AUTHORS Khan.A.A., Khan.M.S., Singh.R. and Raj.S.K. TITLE Direct Submission JOURNAL Submitted (14-JAN-2005) Plant Virology, National Botanical Research Institute, Rana Pratap Marg, Lucknow, UP 226001, India FEATURES Location/Qualifiers source 1 ..770 /organism="Calendulla yellow net begomovirus" /mol_type="genomic DNA" /isolation_source="Calendulla officinalis" /db_xref^"taxon:31I317" /country="India: Lucknow" CDS <1..769 /codon_start=2 /product="coat protein"

Translation="AKRPADIIISTPASKVRRRLNFDSPYVSRAAAPIVRVTKARAWANRPMNRKPRMYRM YRSPDVPRGCEGPCKVQSFESRHDIQHIGKVMCVSDVTRGTGLTHRVGKRFCVKSVYVLGKIWM DENIKTKNHTNSVMFFLVRDRRPVDKPQDFGEVFNMFDNEPSTATVKNVHRDRYQVLRKWHAT VTGGQYASKEQALVKKFVRVNNYVVYNOQEAGKLKIILRMR*WLYMACTHASNPVYATLKIRIY FYDSVTN"

ORIGIN I ggcgaagcga ccagcagata taatcatttc cacgcccgct tcgaaggtac gccgccgtct 61 caacttcgac agcccatatg tgagccgtgc tgctgccccc attgtccgcg tcaccaaagc 121 aagagcatgg gcgaacaggc ccatgaacag aaagcccagg atgtacagga tgtacagaag 181 tccagatgtc cctagaggat gtgaaggccc atgtaaggtc cagtcgtttg agtccagaca 241 tgacattcag catataggta aagtcatgtg tgtcagtgat gttacgcgtg gaactgggct 301 gactcaccga gtgggtaaaa ggttttgtgt taaatccgtt tatgtcttgg gtaagatatg 361 gatggacgag aatattaaga ccaaaaatca cacgaacagt gtgatgtttt ttttagttag 421 ggatcgtaga cctgtggata aacctcaaga ttttggtgag gtgtttaaca tgtttgataa 481 tgagcccagt acggctactg tgaaaaatgt tcatcgtgat aggtatcaag tacttcggaa 541 atggcatgca actgtgaccg gtggacaata tgcgtcaaag gaacaagctc ttgtgaagaa 601 gtttgttagg gttaataatt atgttgtgta taaccagcaa gaagctggca agttgaaaat 661 cattctgaga atgcgttaat ggttgtatat ggcatgtact catgcctcta acccagtgta 721 tgctactttg aagatacgga tctatttcta tgattcggtc acaaattaat