7

2. REVIEW OF LITERATURE

2.1 History of Tomato leaf curl virus (ToLCV) disease

Tomato leaf curl disease is a major constraint in improving the productivity of tomato.

The natural occurrence of a virus associated with the leaf curl disease was observed on

tomato in India by Pruthi and Samuel (1939). Serious nature of leaf curl disease of tomato

was reported in Northern India by Vasudeva and Samraj (1948) and later from Coimbatore

(Ramakrishnan et al., 1964), Delhi (Vasudeva, 1959; Nariani, 1968), Hisar (Varma and

Poonam, 1977; Varma et al., 1986), Karnataka (Govindu, 1964; Sastry and Singh, 1971;

Muniyappa and Veeresh,1984), Kanpur (Singh and Lal, 1964), Kerala (Nair and Wilson,

1969), Lucknow (Srivastava et al., 1975), Maharashtra (Varma, 1959; Mote, 1976; Mayee et

al., 1974; Datar, 1981), Punjab (Butter and Rataul, 1973) and Pantnagar (Saklani and Mathai,

1978). Besides India, tomato leaf curl disease was reported from Sudan (Cowland, 1932),

Srilanka (Shivanathan, 1983), Egypt (Nour Eldin et al., 1969), Philippines (Retuerma et

al.1971), Somalia (Castelloni et al., 1981), Thailand (Tharapase et al., 1983) and Taiwan

(Green et al., 1987). Tomato yellow leaf curl disease (TYLCV) has been reported from Israel

(Cohen and Nitzany, 1966; Nitzany, 1975), Jordan (Makkouk, 1978), Lebanon (Makkouk et

al., 1979) Saudi Arabia (Mazyad et al., 1979) and Thailand (Alathom and Sutabutra, 1986).

Details on the distribution of the Tomto leaf curl disease in different states of India are given

in Table 2.1.

2.2. Symptomatology

Major symptoms of the disease are curling of leaves, enation, yellowing of leaves,

downward bending, reduction of leaf lamina. The older leaves become leathery and brittle.

The disease induces severe stunting, bushy growth, and partial or complete sterility

depending on the stage at which infection has taken place. Infected plants bear few or no

fruit. The pathogen was shown to be transmitted by the whitefly but not by sap inoculation

(Vasudeva and Samraj 1948; Nariani and Vasudeva 1963; Verma et al. 1975; Muniyappa et

al. 1991). It appears to be caused by a complex of several viruses as symptom variations on

different indicator hosts occur (Singh and La11964; Nariani 1968; Reddy et al. 1981). Reddy

et al. (1981) observed various symptoms on tomato. Isolates were divided into five groups:

isolate 1- severe leaf curl with thickening of veins; isolate 2- severe symptoms with enation;

8

isolate 3- screw pattern of leaf arrangement; isolate 4- vein purpling and leaf curl; and isolate

5 -exclusively downward curling of the leaves. Cross protection studies indicated that the five

isolates were all related and were consequently designated tomato leaf curl virus strain 1.

Nariani (1968) reported another strain which was designated as tomato enation leaf curl virus

caused by Nicotiana virus 10A.

2.3. Transmission

The virus is transmitted by the whitefly, Bemisia tabaci gennadius (Hemiptera,

Aleyrodidae) that are attracted to young leaves and growing tips. The virus is not transmitted

mechanically nor via seed. Tomato leaf curl viruses mainly attack tomato, but may also infect

other Solanaceous species. When the populations of B. tabaci are high 90-100% of plants can

become infected resulting in yield loss of 40-100% (Saikia and Muniyappa 1989). Basic

studies on the virus/vector relationship conducted by Butter and Rataul (1977) showed that a

minimum acquisition feeding period of 32 min is required by a viruliferous whitefly to cause

infection on tomato. Preacquisition or preinoculation starving of the vector results in higher

levels of transmission. Females are more efficient in transmission of the disease than males.

The latent period of the virus in the vector is between 21–24 hours. Virus transmission

appears to be affected by temperature, with 33–39°C being optimal. The latent period of the

virus in tomato plants was only 8 days in summer and 90 days in winter (Butter and Rataul

1977). The high incidence of leaf curl disease in the autumn is attributed to the effect of

temperature on virus transmission (Mayee et al., 1974).

2.4. Geminiviruses

Geminiviruses comprise the unique group of viruses characterized by their twinned

particles morphology. These were first described by Goodman in 1977 (Goodman 1977).

Since the earliest report of such structures (Bock et al., 1974; Mumford, 1972; Bock et al.,

1978; Harisson et al., 1977) the members of the family have risen steadily. Morphology of

the virus is unique and geminiviruses have having distinguishing features as: (a) twinned

icosohedral virion (18 x 30 nm) morphology, based on which the family Geminiviridae

derived its name from the latin word ‘geminus’ meaning twin (b) the genome consists of one

or two single stranded circular DNA, of size ranging from 2.5 to 3 kb and (c) the replication

of genomic DNA is through a double stranded intermediate in a rolling circle fashion,

mediated by only one replication initiating protein, Rep. All these features justified the

recognition of the members as a separate family ‘Geminiviridae’ (Murphy et al., 1995).

9

The members of the family cause severe diseases (Rybicki, 1994) of economic

importance, such as the African cassava mosaic disease in the African continent causing

losses upto US $ 1300-2300 million annually (Thresh et al., 1998), Maize streak disease, a

serious threat to cultivation of maize in Africa, and Beet curly top disease, which almost

eliminated sugar beet production from western United States in the early 1900s.

The members of Geminiviridae are divided into four genera (Table 2.2) based on host

range, insect vector and genome organization (Van Regenmortel et al., 2000). The four

genera and their characteristics are given below.

2.4.1 Mastrevirus

The virus species belonging to this genus, have a single genomic component

(monopartite) encoding four proteins (Fig. 2.1a). They infect monocotyledonous plants with

three exceptions, Bean yellow dwarf virus (Liu et al., 1997) from South Africa and Tobacco

yellow dwarf virus (Morris et al., 1992) from Australia and Chickpea chlorotic dwarf virus

from Asia. The type species of genus is Maize streak virus (MSV). The members are

transmitted by leafhopper. The genome, a single stranded circular DNA of 2.5-2.8 kb consists

of four open reading frames (ORFs) and two intergenic, small (SIR) and large (LIR) regions.

Two ORFs in the complementary sense, C1 (Rep A) and C2 (Rep B), together function as

replication initiation protein, Rep. Two virion sense ORFs, V1 (movement protein, MP) and

V2 (Coat protein, CP), encode for cell to cell movement protein and coat protein respectively.

The LIR is similar to the intergenic region seen in other genera, and it is from this region that

rolling circle replication is initiated. Transcription is bidirectional, it starts from LIR and

terminates at SIR (Rybicki et al., 2000).

10

2.4.2. Curtovirus

Members of the genus Curtovirus have monopartite genome (Fig. 2.1b) and are

transmitted by leafhopper in a circulative manner to dicotyledonous plants. The type species

is Beet curly top virus (BCTV) which infects sugarbeet and has a wide host range.

In BCTV, there are seven ORFs among which three are in virion sense (V1, V2 and

V3) and four ORFs in the complementary sense (C1, C2, C3 and C4). The ORF V1 (CP) is

responsible for encapsidation, ORF V2 is involved in regulation of the single stranded (ss)

DNA accumulation and ORF V3 (MP) is responsible for cell to cell movement of the virus.

The complementary sense ORFs are involved in the replication of the virus. ORF C1

functions as replication initiation protein (Rep), C3 as replication enhancer protein (REn)

(Hanley-Bowdoin et al., 1999) and C4 plays a role in the initiation of cell division (Hull,

2002). The ORF C2 acts as the suppressor of PTGS and interacts with the Adenosine kinase

(ADK) protein (Wang et al., 2003).

2.4.3 Topocuvirus

Members of the genus Topocuvirus have monopartite genome and are transmitted by

treehopper to dicotyledonous plants (Pringle, 1999). The genus has only one member, namely

Tomato pseudo-curly top virus (TPCTV) as the type species. The genome of TPCTV shows a

natural recombination between a mastrevirus and an unknown genus (Briddon et al., 1996).

The virus encodes four ORFs in complementary sense named C1, C2, C3 and C4

responsible for replication of the virus and two ORFs in virion sense called V1 and V2

similar to mastreviruses, which are responsible for movement and encapsidation of the virus

respectively (Fig. 2.1c).

2.4.4 Begomovirus

Begomoviruses rank among the top of the most important plant viruses causing

disease of severe consequences in economically and socially relevant crops. The genus

derived its name from the Bean golden mosaic virus. This is the largest genus in the family

and consists of more than 200 recognized distinct virus species and 53 tentative species

(Fauquet et al., 2008). The members exhibit a narrow host range among dicotyledonous

species. The begomoviruses have either monopartite or bipartite genome. Bean golden

mosaic virus (BGMV) is the type species. They are transmitted by the whitefly, Bemisia

11

tabaci Genn. The genome organization of the genus is discussed in detail as it is relevant to

the present study.

Members of the genus Begomovirus are the only geminiviruses to have whitefly as

vector, all of which belong to only one species Bemisia tabaci Genn. Emergence of whitefly

transmitted geminivirus diseases as a major constraint to agricultural productivities in tropical

and subtropical regions of the World (Varma and Malathi, 2003), increased due to many

factors. They are, worldwide increase in the population and distribution of the vector,

extensive monocropping, intensive agricultural endeavour, experimenting with introduction

of new crops in unconventional new areas, new seasons and global movement of plant

materials.

Diseases caused by members of begomoviruses affect the economy of tropical and

subtropical countries (Varma and Malathi, 2003; Rishi, 2004). Typical examples are cassava

mosaic disease in Africa (US $ 2 billion, Thresh et al., 1998), Cotton leaf curl disease in

Pakistan (US $ 5 billion, Briddon and Markham, 2000) and yellow mosaic disease of legumes

(US $300 million annually, taking blackgram, mungbean and soybean together (Varma et al.,

1992). Due to economic consideration, the viruses have received keen attention from research

workers culminating in excellent progress in understanding the gene expression strategies and

the evolutionary trends among these viruses. Given below is a brief review of areas relevant

to the present study.

Molecular cloning, nucleotide sequencing, Koch’s postulate establishment and

genome comparisons of more than 209 begomovirus species (Fauquet and Stanley, 2005)

have led to the clear understanding of the diversity and complexity of begomoviruses. Among

begomoviruses, three types of genome organization are met with (Briddon et al., 2003; Bull

et al., 2004). Some Old World and all the New World begomoviruses have two DNA

components, referred to as DNA A and DNA B (Stanley, 1985; Harrison, 1985; Stanley,

1991; Lazarowitz, 1992; Hanley-Bowdoin et al., 1999; Harrison and Robinson, 1999; Gafni

and Epel, 2002; Mansoor et al., 2003; Briddon et al., 2003; and Rojas et al., 2005). Among

Old World begomoviruses, there are variations in the component. The examples of each type

are given below.

Type I

Begomoviruses having bipartite genome (Fig. 2.2a); genome consists of two, single

stranded DNA components referred to as DNA A and DNA B, of 2.5 to 2.7 kb length. Both

12

DNA A and DNA B are necessary for infectivity and both components are represented in

similar amount within infected tissue e.g. virus species, African cassava mosaic virus

(ACMV), Tomato golden mosaic virus (TGMV) and Indian cassava mosaic virus (ICMV).

Type II

Begomoviruses having monopartite genome; which consists of only one component,

DNA A, which has an organization similar to that of DNA A of Type I (Fig. 2.2b). DNA A

alone is infectious and produces severe typical symptoms on experimental assay hosts and

primary hosts from which the virus is isolated. Species of this type are Tomato yellow leaf

curl virus (TYLCV), Tomato yellow leaf curl Sardinia virus (TYLCSV)

Type III

Begomoviruses having monopartite genome similar to DNA A of Type I virus; along

with it, a satellite DNA component of ~1300 bp length, referred to as satellite DNA β is

present (Fig. 2.2c). DNA A alone is infectious and produces very mild leaf curl symptoms in

experimental assay host, but does not cause typical disease symptoms on primary hosts. DNA

A along with DNA when coinoculated, causes typical symptoms of enation, severe leaf curl

or yellow vein symptom in the primary host. Some species of this type of genome are,

Ageratum yellow vein virus (AYVV), Bhendi yellow vein mosaic virus (BYVMV), and

Cotton leaf curl Rajasthan virus (CLCuRV). The satellite DNA β has no apparent sequence

similarity with the helper virus genome and is fully dependent on the helper virus for

replication and vector transmission (Briddon et al., 2003; Monsoor et al., 2003). The DNA β

has only one ORF named as βC1, which is a pathogenicity determinant and modulator of

symptom development (Saunders et al., 2004). In addition to DNA A and DNA β, a small

DNA molecule of 1300 bp called as Alphasatellite is also present. The alphasatellite

resembles DNA 1 component of Nanovirus.

13

2.5. Genome characterization of begomoviruses

Begomoviruses occuring in Old World have characteristic features distinguishing

them from New World viruses. Most of the New World begomovirus are bipartite whereas

both bipartite and monopartite begomoviruses are present in Old World. The genome of

begomovirus comprises either one or two ssDNA molecules ranging from 2.5 to 3× 103

nucleotides in size (Harrison, 1985). It contains DNA A and DNA B components, which are

necessary for efficient infection. DNA A and DNA B encode genes for different proteins

necessary for viral encapsidation, replication and movement. Table 2.3 provides the names of

open reading frames (ORF) present in DNA A and DNA B. The nomenclature adopted to

refer different ORFs is as given in Van Regenmortel et al. (2003). DNA A is essential for

replication and encapsidation (Rogers et al., 1986; Townsend et al., 1986). In Old World

begomoviruses DNA A, viral sense strand has two ORFs, ORF AV2, pre-coat protein and

coat protein, ORF AV1(Table 2.3). In complementary sense, there are totally five ORFs; two

ORFs, ORF AC1 and AC3, encode replication initiation protein (Rep) and replication

enhancer protein (REn) respectively (Elmer et al., 1988a; Etessami et al., 1991). One more

important ORF is ORF AC2 which activates the transcription of rightward ORFs of both

DNA A and DNA B (ORFs AV1, AV2 and ORF BV1) and so is called as transcription

activator protein (TrAP) (Sunter and Bisaro, 1992). In DNA B, there is one ORF in viral

sense strand coding for nuclear shuttle protein (BV1, NSP) and one in complementary sense

coding for movement protein (BC1-MP) (Lazarowitz, 1992; Frischmuth et al., 1993; Pascal

et al., 1994; Ingham et al., 1995).

2.5.1 Intergenic /common region (IR/CR)

Between the start codon of the leftward and rightward coding regions, there is present

a non-coding intergenic region (IR). Within the intergenic region is a short stretch of 118-200

nucleotide length segment which is near identical in both DNA A and DNA B components of

bipartite begomoviruses and so is called the common region or CR. The nucleotide sequence

of CR/IR is highly specific for a given begomovirus. It contains the following elements.

1. A stem and loop structure: This is common to all geminivirus genome, and also called

as hairpin motif (Arguello Astorga et al, 1994) and is the region where origin of

replication starts. The hairpin contains a GC rich stem and an AT rich loop. The loop

contains a nonanucleotide sequence 5'-TAATATTAC-3’ that is conserved among all

14

geminivirus genome (Rogers et al., 1986; Revington et al., 1989; Lazarowitz et al.,

1992; Orozco and Hanley-Bowdoin, 1998).

2. The second important feature in the IR/CR is the binding site for Rep. Rep is a

sequence specific DNA binding protein that recognizes a 13bp repeat (iterated, iteron)

motif. The repeat element contains two, 5-bp direct repeats (GGTAG) separated by a

central core of 3bp (TAA), GGTAGTAAGGTAG (Bisaro, 1996). These iterons are

considered as Rep binding sites. The number of repeats and the way they are arranged

in the CR is specific for specific lineages of virus.

3. The segment from the tandem repeat of iteron to end of the stem loop is considered to

represent the origin of replications or ori.

4. The promoters of both leftward and rightward genes, TATA boxes and sequences

recognized by various transcription factors are also present in the IR/CR (Hanley

Bowdoin et al., 1999).

2.5.2 ORF AV1 (Coat Protein)

The geminate particle morphology, that gives the name to the family ‘Geminiviridae’,

is the typical feature of members of this family. The coat protein is a fascinating

multifunctional protein that encapsidates one molecule of single stranded circular DNA. It is

required for encapsidation and vector transmission. The viral genome is also protected from

nucleolytic digestion, both inside the plant and the vector.

In all the four genera of the family Geminiviridae there is only one type of coat

protein that forms the twinned structure. The coat protein plays a vital role in regulation of

replication. The disease symptoms are attenuated and the onset of disease is delayed when

plants are infected with the coat protein mutants (Sanderfoot and Lazarowitz, 1996; Unseld et

al., 2004). The coat protein of begomoviruses binds to ssDNA preferentially and co-

operatively (Kirthi and Savithri, 2003; Malik et al., 2005). A putative zinc finger motif

corresponding to amino acid residues 65-85 (in monopartite begomoviruses) was considered

to play a role in DNA binding (Kirthi and Savithri, 2003; Unseld et al., 2004).

The role played by CP in intracellular, cell to cell and long distance movement,

depends on the species and genera of the family. In the case of Mastrevirus, CP plays a role

in nuclear import and export of viral DNA and systemic movement (Liu et al., 1997; Liu et

al., 1999; Liu et al., 2001). In the case of Curtovirus, CP and the ORF V3 bring about

15

systemic infection (Soto, 2001). The most interesting feature is seen among begomoviruses.

Among bipartite begomoviruses there is difference over CP requirement. CP-independent

movement occurs in a well adapted host species and host genotypes wherein the non-virion

forms are transported across the cells. However, in non-permissive species, CP is required

(Wang et al., 1999).

2.5.3 ORF AV2 (Pre-coat protein)

The ORF AV2, overlapping the ORF AV1 in the viral sense strand of DNA A occurs

only in the Old World bipartite begomoviruses and monopartitite begomoviruses. The role of

ORF AV2 in bipartite begomoviruses was examined only in the case of Tomato leaf curl New

Delhi virus (Padidam et al, 1996). Abolishing ORF AV2 expression and expression of

truncated protein resulted in attenuation of symptoms in N. benthamiana (Padidam et al.,

1996). The ORF AV2 mutants showed low level of viral DNA replication in protoplasts

(Padidam et al., 1996). AV2 was also found to be present as monomer, dimer and multimer in

Western blots. Mutation in AV2 also did not affect CP production in infected plants

(Padidam et al., 1996). However, Etessami et al. (1989) reported that the absence of ORF

AV2 did not affect pathogenecity in ACMV. In a monopartite begomovirus, ToLCV-[AU],

inactivation of ORF AV2 led to symptomless systemic infection with reduced titre of all

DNA forms (Rigden et al., 1993). It was shown to function as pathogenicity determinant by

regulation of ss and dsDNA levels (Wartig et al., 1997). DNA-A of bipartite begomoviruses

from the New World lacks an ORF AV2 (Rybicki, 1994; Stanley and Latham., 2005).

2.5.4 ORF AC1 (Replication initiation protein Rep)

Replication initiation protein is the only viral protein absolutely essential for

replication (Elmer et al., 1988a). Rep and REn are predicted to bind simultaneously to the

stem-loop structure and to upstream Rep-DNA complex (Hanley-Bowdoin et al., 1996). It is

present in the nuclei of infected plant cells (Nagar et al., 1995) and plays a key role in

replication and transcription (Laufs et al., 1995). Rep specifically recognizes the viral origin

(Fontes et al., 1994), binds to specific sequences (iterons) found in the CR and cleaves the

phosphodiester bond between the seventh and eighth residue of the conserved nonamer 5'-

TAATATTAC-3' (arrow shows the site of cleavage). Rep binds covalently to 5' phosphate

end and 3' end, remains available for rolling circle replication. After one cycle of replication,

Rep cleaves once again at the newly generated origin sequence. Then rep ligates the nascent

3' end of DNA with the previously generated 5' end releasing an unit-genome length of

16

circular ss DNA molecule (Bisaro, 1996). Thus Rep acts as an endonuclease and ligase as it

initiates and terminates rolling circle replication (Laufs et al., 1995; Orozco and Hanley-

Bowdoin, 1998).

2.5.5 ORF AC2 (Transcription activator protein-TrAP)

The transcription activator protein (TrAP) induces sense promoters in DNA A as well

as in DNA B, at the transcription level (Sunter and Bisaro, 1991; Sunter and Bisaro, 1989).

Sunter (1994) showed that TrAP transactivation was non specific but transactivation by TrAP

homologues in curtoviruses C2 protein was not achieved. TrAP is a zinc binding

phosphoprotein. They bind to ssDNA with the affinity in a sequence non specific manner

(Noris et al., 1996, Sunter and Bisaro, 1992). Three conserved domains have been recognized

in this protein: a basic domain with a nuclear localization signal (NLS) at the N terminus, a

central DNA-binding domain with a non classical Zn-finger motif and an acidic activator

domain at the C terminus (Hartiz et al., 1999). TrAP was shown to activate CP expression by

two distinct mechanisms, i.e., by activation in mesophyll cells and derepression in phloem

cells (Sunter and Bisaro, 1997; Sunter and Bisaro, 2003). The TrAP required for initiating

transcription of the virion sense genes and also involved in suppression of post transcriptional

gene silencing (Vionnet et al., 1999; Vanitharani et al., 2005) and acts as a transactivator of

host suppressor gene (Gopal et al., 2007)

2.5.6 ORF AC3 (Replication enhancer protein-REn)

The ORF AC3 encodes the replication enhaner (REn) protein (~15 kDa) that is

targetted to the nucleus (Selth et al., 2005) and is involved in upregulation of replication by

interacting with Rep and possibly through a role in the recognition of the origin of

replication. Replication enhancer protein (REn) regulates viral DNA accumulation. As

mutations in REn produced delayed onset and attenuation of symptoms (Sunter et al., 1990).

The REn protein enhances viral infection and symptom development, an effect that is due to

its role in induction of viral DNA into replication mode (Elmer et al., 1988a; Sunter et al.,

1990).

2.5.7 ORF AC4 and AC5

The direct or indirect effect of the ORF AC4 in viral replication is not fully

understood. The AC4 protein does not have apparent effect on viral DNA accumulation. In

BCTV, due to the effect of C4 protein, the infected leaves showed downward leaf curling,

vein swelling and enation (Latham et al., 1997). C4 protein in BCTV has also a role in

17

initiation of cell division (Hull, 2002). Recent findings, have implicated the C4 gene product,

to have a role in suppression of silencing (Van Wezel et al., 2002). The ORF possibly has a

role in replication of begomovirus as identified using a yeast model system that supports

MYMIV DNA replication (Raghavan et al., 2004). Mutation analysis of AC4 has shown that

this protein is involved in symptom development.

2.5.8 The DNA B in bipartite begomoviruses

Begomoviruses multiply in nuclei via double stranded DNA in minichromosomes.

Due to their replication in nuclei, geminiviruses need to be exported from nucleus into

cytoplasm and transported from one cell to another.

For bipartite begomoviruses, cell to cell and systemic movement depend on proteins

encoded by DNA B (Brough et al., 1988; Etessami et al., 1988). In bipartite begomoviruses,

DNA B encodes for two proteins, ORF BV1 and ORF BC1 that function in cell to cell

movement in infected plants. The BV1 protein is also termed as the nuclear shuttle protein

(NSP), as it facilitates the movement of the viral genome from the nucleus to the cytoplasm,

whereas the BC1 protein functions as cell to cell, viral movement protein (MP), as it

promotes an increase in the size exclusion limit of plasmodesmata (Sanderfoot and

Lazarowitz, 1996). BV1 localization to nuclei in infected plant as well as protoplast was

shown by immunogold labeling (Sanderfoot and Lazarowitz, 1995; Pascal et al., 1994).

In Bean dwarf mosaic virus (BDMV) the BV1 protein exports viral DNA from the

nucleus to cytoplasm, and BC1 increases the size exclusion limit of plasmodesmata and

mediates the cell to cell movement of viral DNA. Also, in BDMV, both BC1 and BV1

proteins, bind to ssDNA and dsDNA in a form and size specific manner (Rojas et al., 1998).

Coinoculation of ACMV DNA A and DNA B with DNA B mutants containing frame

shift mutation, in either BV1 or BC1 allowed local DNA replication, but did not produce

systemic infection. Moreover, DNA accumulation was reduced in leaves infected with BV1

mutant, but was normal with BC1 mutants (Von Arnim et al., 1993).

The BC1 protein is also a symptom inducing element or pathogenicity determinant in

bipartite begomoviruses and mutation studies of BC1 revealed that the 3’ region of BC1 gene

is associated with symptom development (Duan et al., 1997; Pascal et al., 1994). BC1

proteins of several begomoviruses accumulate in the cell periphery (Lazarowitz and Beachy,

1999; Pascal et al., 1994) to facilitate transport of viral DNA. Zhang et al. (2001) showed that

a short stretch of BC1 amino acids (pilot domain) is responsible for the dichotomy of

18

intracellular location whereas another domain (anchor domain) is necessary for cortical and

nucleus localization.

2.6 Infectivity of cloned components

Majority of the members of the family ‘Geminiviridae’ are not sap transmissible to

the host and it is very difficult to study the viral functions using recombinant clones. In this

context, two methods of delivery of viral DNA are followed to study the pathogenesis. They

are (i) Agrobacterium tumefaciens mediated transfer of the viral DNA (ii) biolistic delivery.

Ti plasmid of Agrobacterium tumefaciens delimited by border sequences of 25 bp is

transferred to the plant cell where it gets integrated with the plant nuclear DNA. Any DNA

between the border sequences is also transferred. This transfer region is exploited to

introduce the cloned viral genome. The process of introduction of viral genome into plants

mediated by the Ti plasmid was termed as ‘agroinfection’ by Grimsley et al. (1986) who used

the technique first to deliver Cauliflower mosaic virus into Brassica napus. Since then the

technique is used in several geminiviruses to study the viral gene functions (BCTV-Stenger et

al., 1991; MSV-Grimsley et al., 1986; WDV-Hayes et al.,1988; BGMV-Morinaga et al.,

1988; ACMV-Morris et al., 1988; MYMIV- Mandal et al., 1997; Chakraborty, 1996; Malathi

et al., 2005; Usha Rani et al., 2004; Biswas and Varma, 2001; MYMV- Jacob et al., 2003;

Karthikeyan et al., 2004; Balaji et al., 2004; PYMV Sung and Coutts, 1995; SLCV

Lazarowitz and Lazdins, 1991; TGMV Elmer et al., 1988b; TYLCV, Kheyr-Pour et al.,

1991; Rochester et al., 1990; Czosneck et al., 1993).

2.7 Satellite DNA β

First satellite DNA with no sequence similarity to the host genome was identified in

tomato plants infected with a begomovirus species, Tomato leaf curl virus (ToLCV) (Dry et

al., 1997). The satellite DNA is 682 nt circular DNA with no open reading frames and no

sequence similarity with DNA of helper virus except for 2 short motifs (1) A nonanucleotide

sequence TAATATTAC and (2) Rep binding motif AATCGGTGTC. The satellite DNA

needed ToLCV genome for its replication, spread and insect transmission but it had no

discernible effects on ToLCV (Dry et al., 1997).

These satellite DNA β molecules required helper virus for its replication, spread and

insect transmission. Saunders et al. (2000) first reported that a DNA β associated with the

Ageratum yellow vein virus forms a disease complex which is responsible for yellow vein

phenotype. Similarly, the cotton leaf curl disease in Pakistan is caused by cotton leaf curl

19

virus in association with satellite DNA β (Briddon et al., 2001). The association DNA β was

shown to cause yellow vein mosaic disease in Okra in India (Jose and Usha, 2003).

The association of satellite DNA β with a bipartite begomovirus, Tomato leaf curl

New Delhi virus was first reported by Sivalingam et al. (2004). Rouhibakhsh and Malathi

(2005) reported that cowpea plants with downward leaf curl, puckering and vein enation with

yellow mosaic symptoms were found associated with DNA β component in addition to DNA

A and DNA B components.

DNA β molecules are symptom modulating sat-DNA, which were identified to be

associated with various monopartite begomoviruses. These molecules are around half the size

(~ 1350 nt in length) of their helper virus. These are ssDNA molecules and rely on the helper

virus for replication, movment in plant tissue and plant to plant transmission by the whitefly,

the vector of begomovirus (Saunders et al, 2000., Briddon et al., 2001).

2.7.1 Genome organization of satellite DNA β

Sequence analysis of full length DNA β molecules revealed characteristic features of

the genome organization. The genome of DNA β consists of a single conserved

complementary sense ORF (βC1), an adenine rich region (A rich region) and a satellite

conserved region (SCR) (Briddon et al., 2003; Mansoor et al., 2003; Zhou et al., 2003). The

satellite conserved region (SCR) is between 199 and 210 nucleotides in length and contains a

predicted hairpin (Stem and Loop) structure with conserved nonanucleotide TAATATTAC.

This region shows a 76-98% sequence identity from different DNA β molecules. There is an

A-rich region of length between 251 and 385 nucleotides and has 48-63% Adenosine content

(Fig 2.2c). It has been proposed that this region of DNA β originated from sequence

duplication to increase the size of the molecule to a quarter or half the size of helper

begomovirus genome. A rich region of DNA β is important for encapsidation and

consequently movement by the helper begomovirus (Bull et al., 2004, Saunders et al., 2000).

The conserved βC1 encodes for a predicted 118 amino acid product with a molecular weight

of 13.7 kDa, except TomLCD β01-LA which has 122 aminoacid and HyVMD β02, has a

truncated 116 aminoacids (Bull et al., 2004). βC1 was found to be a multifunctioned protein

and several roles have been assigned to it. It may function as a suppressor of gene silencing

by counteracting the host defence mechanism (Cui et al., 2005) and act as an essential

pathogenicity protein (Saunders et al., 2004). The suppressor activity as well as symptom

induction requires βC1 nuclear localization (Cui et al., 2005). It was also shown to bind to

20

both ss DNA and ds DNA in a sequence non specific manner, although how this relates to the

function remains unknown.

2.7.2 Functions of DNAβ

DNA β satellite associated with type III begomoviruses infection are required for the

induction of disease symptom in primary host plants (Briddon et al., 2001; Jose &

Ramakrishnan, 2003; Saeed et al., 2005; Saunders et al., 2000; Zhou et al., 2003). DNAβ

molecules can affect the accumulation of helper virus. They may be responsible for either

replication,systemic movement of the helper virus or the suppression of the host defence

mechanism and may provide an appropriate cellular environment for DNA replication

(Saunders et al., 2000). The DNA β may play a direct role in regulation of tissue

differentiation during plant development (Saunders et al., 2004). It has been found that DNA

β can suppress the PTGS (Cui et al., 2005; Kon et al., 2010) and can replace the movement

function of DNA B in a bipartite begomovirus (Saeed et al., 2007).

2.7.3 Diversity among DNA β

The phylogenetic analysis of samples from Africa, the Indian sub continent and

southern China resolved two major groups of satellite DNA β. One group originated from the

host within the Malvaceae and a second from the more diverse group and plants within

Solanaceae and Compositae. There is a relatedness existing within the group on the basis of

host and geography. These findings strongly, support the view of co-adaptation of DNA β

molecules with their respective helper begomoviruses (Briddon et al., 2003). Further analysis

of DNA β from non-malvaceous hosts in east and south East Asia revealed much greater

diversity (Bull et al., 2004).

2.8. Transcript mapping

Information on the transcription start site is essential for the analysis of the promoters

of the gene, which lies immediately upstream of the transcription unit. It also helps to find out

multiple transcription start site for a particular gene. The transcriptional regulations

mechanism involved in gene expression strategies could be elucidated from the studies of

transcript anlaysis.

2.8.1. An overall view of transcript mapping in geminiviruses

Geminiviruses consist of unique group of DNA viruses, are attractive as a model for

studying gene expression, DNA replication and are being used as vectors for gene transfer

21

(Stanley, 1985; Davies and Stanley, 1989). The analysis of the transcripts revealed their

bidirectional mode of transcription and helped in identification and arrangement of common

transcriptional regulatory sequences. The complete details about the transcription start and

end points are also helpful in characterisation of bidirectional promoters of the geminiviruses.

The complete picture of transcriptomes of DNA component(s) of geminiviruses aids in

designing the viral based gene expression/cloning vectors. The transcription start and poly(A)

sites on the geminiviruses has been studied by employing the techniques like Northern blot

hybridisation, S1 nuclease assay, primer extension analysis, RACE PCR and its modifications

and C-PCR (Mullineaux et al., 1993; Townsend et al., 1985; Sunter et al., 1989; Frischmuth

et al., 1991; Accotto et al., 1989; Morris-Krsinich et al., 1985; Shivaprasad et al., 2005). The

information available on geminiviruses about the transcript mapping is limited. The transcript

analysis has been done for Maize streak virus (Morris-Krsinich et al., 1985), Digitaria streak

virus (Accotto et al., 1989), African cassava mosaic virus (Townsend et al., 1985), Tomato

yellow leaf curl virus (Mullineaux et al., 1993), Tomato golden mosaic virus (Sunter et al.,

1989), Abutilon mosaic virus (Frischmuth et al., 1991), Mungbean yellow mosaic virus

(Shivaprasad et al., 2005) and Mungbean yellow mosaic India virus (Usharani et al., 2006).

Rapid amplification of cDNA ends (RACE) is the most popular technique for obtaining full

length cDNA when only part of transcript is known.

I. Mastrevirus

(a) Transcript mapping on Maize streak virus (MSV)

Analysis of poly (A)+ RNA isolated from the infected maize in Northern blot and

sandwich blot using specific probes revealed the virion sense transcripts of MSV whereas it

failed to show transcripts of complementary sense genes due to very low abundance of

transcripts (Morris-Krsinich et al., 1985).

(b) Transcript mapping of Digitaria Streak virus (DSV)

Mapping of DSV viral genome revealed two virion sense RNA species of 1.3 and 1.2

kb in Northern blot. Further analysis of S1 nuclease mapping showed that the 5' terminus of

1.3 and 1.2 kb fragments were at coordinates 0 and 150, respectively and 3' terminus at

approxiamtely coordinate 1060 and further fine mapping results showed the +ve terminus of

the 1.3 and 1.2 kb mRNA at coordinates 27 and 168 respectively. The mapping of

complementary sense genes produced upto five species of RNA in Northern blot and it

22

revealed evidence for splicing in RNA 4- species which was present together with unspliced

RNA 2- species. Both could synthesize two kinds of protein from the same transcription unit.

The extensive overlapping of spliced and unspliced RNA species indicated that splicing

events were temporarily regulated (Accotto et al., 1989).

(c) Mapping of transcripts in Wheat dwarf virus (WDV)

Dekker et al., 1991, analysed the poly (A)+ RNA in Northern blot using virion

specific probe and identified the presence of one species of RNA of 1.1. kb, which was a

1060 nucleotide and resolved by medium resolution S1 nuclease mapping. Further high

resolution S1 nuclease assay mapped the major 5' end of virion sense RNA with C residue at

coordinates 277 and 247 and two lesser abundant RNA at 243 and 248. The 3' terminus of

viron sense transcripts was mapped at 1302 and it was stated that the WDV may use different

mechanisms to express overlapping ORFs V2 and V2 from the same transcript.

The mapping of complementary sense transcripts was unsuccessful due to their low

abundance. But 3' RACE PCR mapped the 3' end of spliced and unspliced RNA. Further

results showed that 22.6 % of the cDNA were derived from RNA spliced between n.t.

coordinates 1968 and 1976, splicing out an 86 bp intron. Primer extension analysis employed

for 5' terminus mapping of two prominent RNA species corresponded to nucleotide

cordinates 2690 and 2693 on the WDV genome and a multiple bands, was mapped between

30-40. The presence of multiple overlapping transcripts may suggest temporal regulation of

transcript.

II. Begomoviruses

(a) African cassava mosaic virus (ACMV)

Northern blot analysis of poly(A)+ RNA isolated from ACMV infected plants, probed

with ds DNA A, showed a majority of RNA bands of 1 kb and two other RNA(s) of 1.7 kb

and 0.7 kb (Townsend et al., 1985). S1 nuclease assay with full length probes of ss DNA of

either orientation of DNA A revealed two fragments of 0.6 kb and 1.5 kb with virion sense

probe and a large quantity of 1 kb poly(A)+ RNA with complementary sense probe. Further

mapping of the 1 kb transcript of DNA A coding for a longer ORF in virion sense showed its

5' and 3' termini near 28 and 1240 respectively. The size, orientation and origin was found

from the hybridization with ss DNA. It was identified that 1.7 kb and 0.7 kb RNA were

specific to the complementary sense and code for overlapping ORFs.

23

The poly(A)+ RNA northern blot probed with ds DNA B (DNA 2) detected two

major bands of 1.1 kb and 0.9 kb and a minor band of 1.35 kb. Further, S1 nuclease

protection assay done with probe of ss DNA B (DNA 2) of either orientation revealed virion

sense 1.0 kb band 0.8 kb and small quantity 1.3 kb fragments of complementary sense. RNA

of 1.1 and 0.9 kb were the transcripts of virion sense and complementary sense of DNA B,

respectively. However, the 1.3 kb transcript detailed might represent a precursor of the 1.1 kb

RNA in DNA B component.

(b) Abutilon mosaic virus (AbMV)

Transcript mapping in AbMV by Frischmuth et al. (1991) showed that transcription is

bidirectional and the transcripts were polyadenylated. Northern blots detected a 0.9 kb viron

sense, two overlapping transcripts of 1.6 and 0.7 kb to the complementary sense strand of

DNA A whereas DNA B had a 1.0 kb virion sense, and two 1.3 and 1.2 kb complementary

sense transcripts. Tentative location of these transcripts was determined by S1 nuclease

mapping. Further, results of primer extension analysis to define the 5' terminus of transcripts

revealed that the transcripts of AC1,AV1, BC1 and BV1 starts in or close to the common

region whereas AC2 starts upstream of ORF AC2. Transcript analysis for ORF BC1 yielded

several additional faint bands indicating several 5' ends for this ORF. The ORF AV1

transcripts 5' end mapping, showed multiple bands with upto six nucleotide variations in

primer extension analysis.

For most of the six ORFs transcripts start with A residue, the most frequent base seen

in the beginning of the plant mRNA (Joshi, 1987a). The transcripts of BC1 and BC2 were

overlapping and were similar to that of ACMV and TGMV (Townsend et al., 1985; Sunter

and Bisaro, 1989).

Reverse transcription and PCR analysis to determine the 3' end revealed a single

polyadenylation site for transcripts at AV1, BC1, BV1 and BC2 whereas AC1 and AC2

exhibited some heterogenicity in termination. The 3' ends of the transcripts were found to

overlap in a relatively small region which contained several polyadenylation signal motifs.

Most of the transcripts were found to end at an expected distance of 18-30 bp from a full

consensus sequence AATAAA for plant mRNA (Joshi, 1987b). The heterogenicity or

multiplicity of termination sites might be a means of enhancing the efficiency of

polyadenylation (Frischmuth et al., 1991).

(c) Tomato golden mosaic virus (TGMV)

24

Sunter et al. (1989), analyzed the poly(A)+ RNA isolated from TGMV infected N.

benthamiana leaves by Northern blots, detected one major RNA of 860 nts and three

complementary RNA of 1640, 1040 and 710 bp size, using DNA A components negatively (-

ve) and positive (+ve) strand probes, respectively.

The TGMV DNA B probes of +ve and -ve strands identified each RNA species of

1330 nt in complementary sense and 920 nts of virion sense, respectively. Further internal

probes of specific ORF(s) of DNA A and DNA B revealed the same results and gave an idea

that the complementary transcripts of the DNA share a common 3' terminus. In addition, low

resolution S1 nuclease assay was done to determine the accurate size of viral transcripts.

High resolution S1 nuclease analysis to map the precise 5' terminus of AR1/AV1,

revealed that the 5' terminus was between 316 and 321, reflecting heterogenicity in

transcription start site or imprecise digestion of DNA/RNA hybrid by S1 nuclease assay.

Further, the primer extension analysis, gave two major and three minor products. The two

major bands represented the RNA with 5' start site at nt 319 and 320, in agreement with the

S1 nuclease assay. The minor bands reflected that there may be an existence of multiple

transcription start site based on results of premature RT reaction (or) template cleavage by

RNase (or) combination of both. The major projected 3' terminus of AV1 by S1 nuclease

assay was at near nt 1091. The small diffuse bands observed were probably digestion

products of DNA/RNA hybrids at A-T rich region surrounded at nt 1080.

(d) Tomato leaf curl virus (ToLCV)

The transcript analysis of the monopartite virus ToLCV DNA A component was

carried out by Mullineaux et al. (1993).With the help of S1 nuclease mapping, it was

calculated that the 5' end of ORF V1 RNA was at nt coordinate 133 close to BamHI site and

the 3' end was at the coordinate 1142. Further, precise mapping of 5' end using RNase A/T

protection procedure indicated two 5' termini at coordinates 143 and 175, respectively. An

altenative method of precise mapping using 5' RACE PCR produced a smear of bands due to

heterogenous length of poly(dt) tail addition in the procedure. This result revealed two

clusters of 5' ends close to those determined by RNase A/T protection assay. 3' RACE

procedure to determine the 3' end of the virion sense transcript, identified from the majority

of the clones sequenced, was at coordinate 1095 (Mullineaux et al., 1993).

25

S1 nuclease mapping was unsuccessful in the case of complementary sense transcripts

because of the low abundance of transcripts. 5' RACE PCR analysis indicated clustering of 5'

ends at coordinates 2646 and 1655/1656 against single clones whose 5' ends were at different

nt coordinates. But the 3' end for all complementary sense ORFs by 3' RACE revealed the

predominance of only one 3' end.

RNase A/T1 mapping results for apparent 5' end 2646 identified, one 5' termini of

2644 which was upstream of ORF C1 and close to the cluster of 5' termini identified by 5'

RACE whereas two other 5' termini at 2715 and 2554. The RNase A/T mapping for the 5' end

of transcript coordinate 1656, revealed two 5' end for ORF C2 at nt coordinate 1660 and

1663, close to the position mapped by 5' RACE PCR (Mullineaux et al., 1993).

(e) Mungbean yellow mosaic virus (MYMV)

Shivaprasad et al. (2005) adopted circularization RT-PCR (cRT-PCR) for the

simultaneous mapping of both the transcription start site (cap site) and the polyadenylation

site of a given mRNA. The major rightward transcription initiation (TSS) of virion sense

ORF(s) on DNA A were mapped to positions A 137 and A 141, generating two distinct

mRNAs. The RNA start site at A 137 could be used for translation of the ORF AV1 but not

of AV2 due to lack of necessary leader sequence. The complementary sense ORF(s) TSS

were mapped at A 2649 for AC1/4, at A 1649 and A 1646 for AC2/3, respectively.

Mapping of poly(A) sites for rightward transcription unit had a single major poly(A)

site at A 1095, which was 22 nt downstream of the poly(A) signal, whereas for

complementary ORF(s) of DNA A, a single common poly(A) site was identified at A 1066,

located 5 nt downstream of the AC3 ORF and 21 nt downstream of the near poly(A)

consensus signal (AAUACA).

The transcription start site for the virion sense ORF BV1 was mapped at two major

positions, A 410 and A 414 at an optimal distance from the TATA box and five nt upstream

of BC1 AUG start (at 419) where the complementary sense ORF BC1 transcription start site

was identified at A 2359, just 33 bp upstream of the non consensus TATA box signal

(TATTTAAA).

The 3’end mapping results of ORF BV1 showed a total of 12 poly(A) sites from A

1191 to G 1287 and it was found that the 3’end processing of ORF BV1 was very imprecise.

It was also observed in the case of leftward transcription unit BC1, in which the poly(A) site

26

was distributed over a 40 bp region and revealed multiple termination sites of transcripts. In

addition, BC1 transcription unit analysis, also identified a 123 nt intron within the transcript

of BC1 which could spliced to yield a processed transcript.

Northern blot analysis of total RNA from blackgram infected by MYMV identified

transcripts of 1.65 kb AC1/4 transcript (by AC1 and AC2 probes), 0.65 kb AC2/3 transcript

(using AC2 probe) 1.0 kb AV2/1 transcript (by AV1 probe), 0.9 kb BV1 transcript (BV

probe), 1.1 kb spliced BC1 transcript (BV probe), 1.1 kb spliced BC1 transcript, and three

additional less abundant transcripts of ~ 0.7 kb, ~ 1.2 kb and ~ 1.4 kb (probe BC1). The ~ 1.2

kb transcript was likely to be an unspliced version of the BC1 transcript (Shivprasad et al.,

2005).

2.8.2. Transcript mapping in satellite DNA β

The transcripts of ORF βC1 of satellite DNA β associated with ageratum yellow vein

disease was mapped by Saunders et al. (2004). The Northern blot analysis using poly (A)

RNA extracted from infected plants showed a abundant DNA β specific transcript of βC1 by

probe βC1 but not other regions of DNA β. Transcript mapping of βC1 transcript by 5' RACE

mapped the 5' termini at A 555, located eight nt upstream of AUG start codon and 34 nt

downstream of the consensus TATA box. 3’RACE analysis indicated multiple 3' termini in

the downstream of ORF βC1 between 68 and 157 position and position 128 being most

frequently observed.

2.9 Promoters

Promoter is a DNA sequence normally located upstream of the transcribed region. It

contains a TATA box and serves to determine the start site of transcription (Dynan and Tjian

1985). Transcription factors together with RNA polymerase recognize a promoter region by

its structural features and associate with it to initiate transcription. In this process, the newly

formed complex positions RNA polymerase at the transcription initiation site and activates

transcription (Lewin, 2008). Promoters typically have a modular structure, consisting of

multiple short sequence (5 to 20 nucleotides), called cis-acting regulatory elements, most of

which comprise transcription factor (TF) binding sites. These elements can be dispersed or

can overlap and usually lie within the 1kb region upstream and surrounding a transcription

27

start site (TSS). The combination of these regulatory elements is often unique for most genes

involved in various pathways. The promoter can roughly be divided in two parts: a proximal

part, referred to as the core, and a distal part. The proximal part is believed to be responsible

for correctly assembling the RNA polymerase II complex at the right position and for

directing a basal level of transcription (Nikolov et al., 1996; Nikolov and Burley, 1997; Berk,

1999; Rombauts et al., 2003). It is mediated by elements, such as TATA and initiator boxes

through the binding of the TATA box-binding protein, and other general TFs (Featherstone,

2002). The distal part of the promoter contains elements that regulate the spatio-temporal

expression (Tjian and Maniatis, 1994; Fessele et al., 2002). In addition to the proximal and

distal parts, somewhat isolated, regulatory regions have also been described, mainly in

animals that contain enhancer and/or repressors elements (Barton et al., 1997; Bagga et al.,

2000). The latter elements can be found from a few kilobase pairs upstream of TSS, in the

introns or even at the 3’ of the genes they regulate (Larkin et al., 1993; Wasserman et al.,

2000).

Depending on the activity, promoters can be classified as constitutive, tissue-specific

and inducible. A constitutive promoter contains elements recognized by basal activators or

transcription factors to initiate transcription in all tissues at all times. However, inducible

promoters are activated by one or more stimuli such as hormones (auxin, abscisic acid,

gibberellic acid, ethylene, salicylic acid and/or methyl jasmonate), chemicals (tetracycline,

dexamethasone, copper, nitric oxide), environmental conditions/stresses and biotic stresses,

whereas tissue-specific promoters control gene expression in a tissue-dependent manner and

according to the developmental stage of the plant.

Constitutive promoters are the most common promoters used to drive the expression

of various genes in plants. The most commonly used promoter for directing strong

constitutive expression has been the Cauliflower Mosaic Virus (CaMV) 35S promoter (Odell

et al., 1985; Jefferson, 1987). Although a number of constitutive promoters have been

isolated from plants and used for the generation of transgenic plants, most of them are

protected by patents. Therefore, novel plant sequences that can function as promoters for the

high-level expression of transgenes are to be identified and tested. A wider range of effective

promoters would also make it possible to introduce multiple transgenes into plant cells while

still avoiding the risk of homology-dependent gene silencing (Schunmann et al., 2003). Large

number of promoters were isolated from begomoviruses. In the case of Tomato yellow leaf

curl china virus (TYLCCNV) 173 nt fragment upstream of βC1 was found to have phloem

28

specific expression (Guan & Zhou, 2006) whereas in Cotton leaf curl Multan betasatellite the

68nt region upstream of βC1(-207 to -139) was having strong promoter activity (Eini et al.,

2010). Besides betasatellite, the intergenic region present in helper virus DNA-A has a

potential promoter activity. Intergenic region of geminiviruses possess a bidirectional

promoter which mediates the transcription of CP and Rep genes (Sunter and Bisaro, 1997,

Xie et al., 2003). CP promoter from Tomato golden mosaic virus (TGMV) is active in both

phloem and mesophyll cells in the presence of a transcriptional activator protein (TrAP).

Complementary promoter of Rep gene was found to have stronger activity than virion sense

promoter in the case of African cassava mosaic virus (ACMV) (Zhan et al., 1991) and Cotton

leaf curl Multan virus (CLCuMV) (Xie et al., 2003). CP promoter from Maize streak virus

(MSV) was found to be active in vascular tissues of transgenic rice plants. Based on

histochemical staining it is proved that wheat dwarf virus viral sense promoter can induce an

expression pattern specifically within the phloem cells (Dinant et al., 2004) on the basis of

transient expression of GUS gene.

2.10 RNA Interference (RNAi)

RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a

biological response to double-stranded RNA that mediates resistance to both endogenous

parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-

coding genes. This natural mechanism for sequence-specific gene silencing promises to

revolutionize experimental biology and may have important practical applications in

functional genomics, therapeutic intervention, agriculture and other areas.

RNAi pathway include endogenous triggers of foreign DNA or double-stranded RNA

(dsRNA) of viral origin, aberrant transcripts from repetitive sequences in the genome such as

transposons, and pre-microRNA (miRNA). In plants, RNAi forms the basis of virus-induced

gene silencing (VIGS), suggesting an important role in pathogen resistance. RNA silencing is

an innate antiviral defense system in the plants which involves sequence specific degradation

of viral RNA. In plants double stranded replicative intermediates of RNA viruses or

overlapping structured segments of mRNA transcripts of DNA viruses are the targets, which

are processed by DCL enzymes into short 21nt dsRNA having 3’ overhangs called viral

siRNAs (Voinnet et al. 2001). These siRNAs bind to nuclease complex known as RNA

induced silencing complex (RISC) guiding the complex to bind and destroy homologous

transcripts (Hammond et al. 2000).

29

RNAi pathway involves the long double stranded RNA. A simplified model for the

RNAi pathway is based on two steps, each involving ribonuclease enzyme. In the first step,

the double stranded RNA (dsRNA) is processed into short (21-25 nucleotide) interfering

RNA (siRNA) by the RNAase III Dicer enzyme which exist in a double stranded form with

two nucleotide overhangs at the 3’ ends. In the second step, siRNAs are loaded into the

effector complex RNA-induced silencing complex (RISC). The siRNA is unwound during

RISC assembly and the single-stranded RNA hybridizes with mRNA target. Argonaute

protein (AGO) is a core protein and exhibits structural similarity to RNase H (Pendon and

Ding 2008). They bind siRNA (Song et al., 2003, Ma et al., 2004) and are the “slicer”

ribonucleases that degrades the target RNA (Liu et al., 2004; Meister et al., 2004; Rand et al.,

2004). Small RNAs guide Argonaute proteins to their specific targets through sequence

complementarities, which typically leads to silencing of the target. Some of the Argonaute

proteins have endonuclease activity directed against messenger RNA (mRNA) strands that

display extensive complementarity to their bound small RNA, and this is known as Slicer

activity (Tolia & Joshua et al., 2007). Ten homologues of AGO (1–10) are found in

Arabidopsis thaliana and mainly function as ‘slicers’ in this pathway (Chapman and

Carrington 2007). Binding of siRNA and miRNA into RISC requires a specific dsRNA

binding protein (DRB) that hetrodimerizes with Dicer. Plants have four types of Dicer like

(DCL) enzymes, each produces small RNAs with different functions. DCL-1 produces

microRNAs (Park et al., 2000) which can mediate either cleavage or translational repression

of the target mRNA in an AGO dependent manner. DCL-2 has the ability to perform

sequence specific endonucleolytic cleavages of long dsRNA DCL-2 generates both stress

related natural antisense transcript short interfering RNAs (siRNAs) (Borsani et al; 2005) and

siRNA against African cassava mosaic virus (ACMV) Cabbage leaf curl virus (CaLCuV)

(Xie et al 2004; Akbergenov et al., 2006), DCL3 produces approximately 24 nt siRNA that

direct hetrochromatin formation (Xie et al., 2004) and DCL4 generates both trans acting

siRNAs, which regulate some aspects of developmental timing, and siRNAs involved in

RNAi (Dunoyer et al., 2005; Gasciolli et al., 2005; Xie et al., 2005). Both DCL-2 and DCL-4

process long dsRNA sequentially, starting from one end.

In recent years, a major breakthrough in the study of RNA silencing in plants came

with the discovery that RNA silencing is generally non-cell autonomous (Hyun et al., 2011).

Plants can generate extensive RNA silencing through non-metabolic, gene-specific diffusible

signals with sequence-specific information that travel from cell to cell through

30

plasmodesmata (channels that connect most plant cells) (Lucas et al., 2009) and over long

distances through the vascular phloem (tissue that distributes nutrients throughout the plant)

to different organs (Voinnet et al., 1998; Mlotshwa et al., 2002; Himber et al., 2003; Voinnet,

2005). Non-coding small RNAs (miRNAs, siRNAs) play diverse and inherently integrative

roles in the cellular system

2.10.1. Geminiviruses and RNA silencing

In plants gene silencing phenomenon based on co-suppression and was first

discovered in transgenic petunia when an attempt to overexpress chalcone synthase in petunia

plants by introducing a chimeric petunia CHS gene resulted in loss of expression of both the

transgene and the homologous endogenous gene (Napoli et al., 1990). This phenomenon is

known as quelling in fungi (Cogoni & Giuseppe, 1997) and RNA interference in animals

(Fire et al.,1998) that has common steps in these organisms (Pickford & Cogoni, 2003). RNA

silencing pathways are involved in cellular defense against viruses, controlling transposon

mobility, development of the organism by microRNAs, histone and DNA methylation, and

establishment of hetrochromatin (Baulcombe 2004; Vionnet 2005).

Three silencing pathway have been described in plants including PTGS a cytoplasmic

gene silencing which is important for virus protection, transcriptional gene silencing

associated with DNA methylation and suppression of transcription that can protect the

genome from transposons and microRNA regulating gene expression by silencing of

endogenous messenger RNAs which have a key role in plant development (Baulcombe,

2004).

Plant viruses can be targets of RNA silencing in plants. This is an important

mechanism of host defense against viruses. This phenomenon was revealed by the finding

that the synergism between viruses in which symptom severity of a virus disease was

increased by coinfection with an unrelated virus (Vance et al., 1995). In addition recovery

from disease can occur both in DNA viruses such as Cauliflower mosaic virus (CaMV) which

replicate in the nucleus and RNA viruses such as Tobacco rattle virus, which replicate in

cytoplasm (Al Kaff et al., 1998; Lecellier et al., 2004; Vaucheret & Fagard, 2001).

Furthermore detection of virus specific siRNA in plants infected with RNA viruses as well as

silencing suppression proteins such as helper component proteinase (HcPro) and 2b that is

described as a pathogenicity determinant, provides other evidence that plant viruses

commonly induce an RNA silencing response (Hamilton & Baulcombe, 1999; Patrice et al;

31

2004). Finally mutants of some essential genes such as transgene induced silencing such as

RdRP6 also known as SDE1/SGS2 and required in S-PTGS, SDE3 and AGO1 enhanced the

susceptibility of Arabidopsis to viral infection (Ding et al., 2004).

Virus infection is sufficient to induce a PTGS like response in the absence of

sequence homology between viruses and host nuclear genes (Lecellier et al., 2004). It seems

that the dsRNA produced as a replication intermediate for RNA viruses or some viral ssRNA

with high secondary structure elicits this protection response. Following virus infection,

nuclear transgenes homologous to viral sequence became methylated suggesting that viral

RNAs present in the cytoplasm entered the nucleus and triggered DNA methylation. In

addition, RdDM has also been observed in silenced tissue infected with cytoplasmically

replicating RNA viruses (Hamilton et al., 2002; Jones et al., 1998). RNA silencing has been

reported for CaMV a dsDNA virus. Several siRNAs produced from this virus were shown to

be implicated in regulation of host genes (Moissiard & Voinnet, 2006).

Geminiviruses can both induce and be targets for gene silencing (Vanitharani et al.,

2005). Production of siRNA and PTGS have been reported from both monopartite (Lucioli et

al., 2003) and bipartite geminviruses (Chellappan et al., 2004). There was a significant

correlation between recovery phenotype, siRNA production and the level of viral DNA and

mRNA in ACMV infected plants (Chellapan et al., 2004). Geminiviruses are targeted by all

four DCL activities to produce three major sizes (21, 22, 24 nt) of viral siRNA from both

coding and noncoding regions for DNA viruses. DCL4 and DCL3 were more important for

production of siRNA in plants infected with DNA viruses (Blevins et al., 2006). These

siRNAs are methylated at the 5’end and phosphorylated at the 3’ end (Akbergenov et al.,

2006). Geminivirus do not have dsRNA replicating form in their disease cycle yet they can

induce the PTGS in infected plants. The dsRNA in geminiviruses can be produced in three

possible ways as bidirectional transcription (Townsend et al., 1985) in which viruses produce

polycistronic mRNA with opposite polarity from the conserved region that overlaps at the

3’end and results in the formation of dsRNA. Supporting this possibility the majority of

siRNA in ACMV-[CM] infected plants are derived from N-terminus of the AC2 which

overlap the C-terminus of AC1 (Challapan et al., 2004). Alternatively the host RdRP can

possibly use the C1 transcript as a template to produce dsRNA (Dalmay et al., 2000) without

using an exogenous primer (Tang et al., 2003) or it can extend the overlap between the two 3’

ends of the mRNAs. However DNA viruses do not code for an RdRP and it has been shown

that mutation of the host RdRP2 and RdRP6 are also dispensable for the biogenesis of

32

siRNAs from DNA viruses (Blevins et al., 2006).Another possibility is induction of hairpin

structures in the geminivirus transcript that can be targeted by the DICER to produce siRNA

(Challapan et al., 2004).

2.10.2. Suppressors of RNA silencing

Majority of the plant viruses have evolved with suppressor proteins to evade this

defense mechanism of the plant (Roth et al., 2004; Voinnet et al., 1999). Distinct suppressor

proteins encoded by the members of different plant viruses suggest that plant viruses evolved

this counter defensive mechanism independently on many occasion (Tijsterman et al., 2002;

Vaucheret et al., 2001; Dunoyer et al., MacDiarmid et al., 2005). RNA silencing suppressors

from different plant viruses are structurally different. Many suppressors function as the

pathogenicity factors that cause the developmental abnormalities (Cui et al., 2004; Lindbo et

al., 2005). Viral suppressors are supposed to function at distinct steps of silencing machinery.

Begomoviruses produce the three different suppressor proteins as AC4, AC2 and βC1. AC2,

which encodes the transcriptional activator protein (TrAP) of African cassava mosaic virus-

[Kenya] Tomato golden mosaic virus and Mungbean yellow mosaic virus [Vigna] is a

positional homologue of C2 in Tomato leaf curl virus-[Australia] and Tomato leaf curl China

virus (TYLCCNV) exhibit sequence non specific DNA binding activity and confined to

nucleus (Randles et al., 2004; Trinks et al., 2005; Voinnet et al., 2000). For the monopartite

begomoviruses, the AV2 protein of TYLCV-IL has recently been identified as an RNA-

silencing suppressor and may exert its suppressor effect by targeting a step in the RNA-

silencing pathway that occurs after siRNA production (Avi et al., 2007)

2.10.3. Begomovirus defense mechanism against RNA-silencing of plants

Many suppressor proteins were encoded by different viruses which are as given.

2.10.3.1. Cucumber mosaic virus (CMV) 2b:

The CMV 2b protein was one of the best studied and first identified suppressor of

RNA silencing. Stable expression assays demonstrate that the 2b protein encoded by CMV

gives partial intracellular suppression of silencing in association with reduction of siRNA

accumulation and inhibition of methylation of siRNA targeted transgenes (Elmayan and

Vaucheret., 1996; Guo and Ding 2002). Grafting experiments and transient expression

experiment shows that 2b blocks the movement of systemic silencing signal.

2.10.3.2. Potyviral helper component protease (HC-Pro):

33

HC-Pro was the first identified suppressor of RNA silencing. All the previous reports

demonstrated that it suppresses both transgene and virus induced silencing (Anandlakshmi et

al., 1998; Kasschau and Carrington, 1998). As compared with CMV 2b protein, it is able to

reverse established silencing in the reversal of silencing assay (Brigneti et al., 1998) suggests

that these two suppressors as HC-Pro and CMV 2b work at different steps in the silencing

pathway. It interferes with the initiation and maintenance of silencing at a step coincident

with upstream of siRNA production, because it did not prevent the silencing signal from

becoming systemic (Llave et al., 2000).

2.10.3.3. Tombusvirus P19:

P19 protein is a suppressor of silencing that is extremely potent in the transient

expression assay in which it blocks the both intracellular and intercellular RNA silencing. It

is supposed to be a weak suppressor, only reversing silencing in the regions of veins. In

transient expression assays P19 blocks both local and systemic silencing and eliminates all

small RNAs (Hamilton et al., 2002, Voinnet et al., 2003). Biochemical studies have shown

that P19 binds siRNA and that binding depends on characteristics of RNase III products

(dsRNAs with two nucleotide 3’ overhangs) (Silhavy et al., 2002). This result reveals that

P19 suppresses silencing by sequestering siRNAs, thereby preventing their incorporation into

the RISC complex to serve as guides. This is a novel mechanism among suppressors and

because it theoretically stems from an intrinsic property of the protein to bind functional

siRNA.

2.10.3.4 Potato virus X (PVX) p25:

Protein p25 is known to be required for cell to cell movement of potexviruses.

Agroinfiltration experiments showed that p25, without any other PVX protein was sufficient

to block systemic silencing. Theses transient expression experiments make a convincing case

that PVX p25 blocks the movement of mobile silencing signal.

The recent development of molecular techniques has led to significant advances in our

knowledge of begomoviruses, their genomes and roles in disease etiology (Sharma &

Ikegami, 2008). Begomoviral encoded suppressor proteins are believed to act at different

points in the silencing pathways. Considering that begomoviruses replicate in the nucleus and

their genomes are made up of DNA and do not possess a dsRNA phase in their replication

cycle, how do they then trigger RNA silencing in plants? The begomovirus dsDNA serves as

the template for both replication and transcription, with the transcription occurring

34

bidirectionally with two major polycistronic transcripts in opposite orientation from the CR

(bipartite begomoviruses) or IR (monopartite begomoviruses) that contains the bidirectional

promoter sequences. The AV2-AV1 (bipartite begomoviruses)/V2-V1 (monopartite

begomoviruses) transcripts and the complementary sense AC1-AC3(bipartite

begomoviruses)/C1-C3(monopartite begomoviruses) transcripts overlap by 4 nts at their 3’

ends (Chellappan et al., 2004). It is therefore thought that the overlapping transcripts in

opposite polarity at the 3’end may generate double-stranded mRNA (dsmRNA), which could

induce RNA silencing (Voinnet, 2001) and are therefore targets of RNA-silencing in plants

(Muangsan et al., 2004). In bipartite begomoviruses, the AC2 (TrAP) has been reported to

suppress RNA-silencing by controlling the expression of host genes coding for positive or

negative effectors of RNA-silencing. It is believed to inactivate adenosine kinase (ADK),

whose function has been implicated in the methylation of the replicative form of

begomoviruses and therefore suppresses local silencing (Trinks et al., 2005; Vanitharani et

al., 2005; Wang et al., 2005). The C4 protein of bipartite begomoviruses such as ACMV,

EACMV, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus

(SLCMV) can suppress RNA silencing due to their ability to bind to micro RNA (miRNA)

and small interfering RNA (siRNA) (Vanitharani et al., 2004; Chellappan et al., 2005;

Fondong et al., 2007). For the monopartite begomoviruses, the V2 protein of TYLCV-IL has

been identified as an RNA-silencing suppressor (Table 2.4) and may exert its suppressor

effect by targeting a step in the RNA-silencing pathway that occurs after siRNA production

(Avi et al., 2007). It is therefore thought that TYLCV-IL encodes two types of RNA-

silencing suppressors the V2 protein for earlier silencing events and C2 protein for the later

silencing events (Zrachya et al. 2007). Betasatellites that associate with monopartite

begomoviruses have been shown to induce typical disease symptoms in plants and suppress

gene silencing (Saunders et al., 2000; Briddon et al., 2001; Mansoor et al., 2003; Briddon et

al., 2003; Cui et al., 2005; Kon et al., 2006). It has been demonstrated by mutational analysis

that the betasatellites single ORF encodes the pathogenicity determinant βC1 and transgenic

expression of the 14 kDa βC1 protein or expression from a Potato virus X (PVX) vector

results in severe developmental abnormalities (Zhou et al., 2003, Cui et al., 2004; Saunders et

al., 2004; Kon et al., 2007; Tao and Zhou, 2008; Yang et al., 2008). Cui et al. (2005) had

shown that TYLCCV βC1 functions as the suppressor, binds non-specifically to DNA,

suppress the PTGS and is targeted to cell nucleus. The precise mechanism of action of the

βC1 protein is presently unknown.