Keywords

9.1 Introduction

Geminiviruses (family Geminiviridae) are a large group of virus es infecting plants and are responsible for causing crop losses worldwide and are transmitted by insect vectors (white flies). The geminivirus genome is a single-stranded DNA which is encapsidated by twin incomplete icosahedral particle. There are seven genera in the family Geminiviridae: Begomovirus, Becurtovirus, Curtovirus , Eragrovirus, Mastrevirus , Topocuvirus , and Turncurtovirus based on their host range, insect vector , and genome organization (Varsani et al. 2014). The genus Begomovirus have emergent pathogens of crops throughout the tropical and subtropical regions of the world. Continuous emergence of begomoviruses is a threat to vegetable production in Southeast Asia (Varma and Malathi 2003; Varma et al. 2011, 2012, 2013). Begomovirus is the largest genus of plant viruses with respect to the number of species (288) presently recognized by the International Committee on Taxonomy of Viruses (ICTV) (Brown et al. 2015). Whitefly-transmitted geminiviruses cause tremendous losses in the number of vegetable and cereal crops throughout the American and the Caribbean Basin, the Mediterranean Plain, India, and Southeast Asia. Various crops are affected by begomoviruses such as cassava, cotton, bean, pepper, and tomato (Brown et al. 2015; Varma et al. 2011). In the Old World (OW; Africa, Asia, Australia, and Europe), mostly monopartite, begomoviruses are present with very few having a bipartite genome in comparison to begomoviruses native to the New World (NW; the Americas) which are almost exclusively bipartite, with only a single monopartite virus identified so far. However, a number of monopartite begomoviruses do occur in the NW as well as a result of their introduction from the OW (Sánchez-Campos et al. 2013; Melgarejo et al. 2013). New World and Old World begomoviruses are different genetically, and they fall in a separate group in phylogenetic analyses. The OW viruses show a greater genetic diversity and have an additional, conserved gene (known as V2 for the monopartite and AV2 for the bipartite viruses) which is absent in the NW begomoviruses.

Geminiviruses have monopartite genomes except begomoviruses. The begomovirus genome is bipartite (two DNA components; DNA-A and DNA-B, 2.6 kb) or monopartite (similar to DNA-A, 2.8 kb). With a few exceptions (Albuquerque et al. 2011; Choi et al. 2012), there is always association of betasatellite or alphasatellites or both in monopartite begomoviruses (Briddon et al. 2003). There are some reports available where satellites have also been found to be associated with bipartite begomoviruses (Romay et al. 2010; Jyothsna et al. 2013; Sivalingam and Varma 2012).

Till date two satellite DNA molecules associated with begomoviruses are reported: betasatellite and alphasatellite. Earlier known as DNA-β and now called as betasatellites, these molecules are found associated with monopartite begomoviruses, ~1360 nt in length (half the size of the helper virus genome). These betasatellites do not have any similarity in sequence with the helper viruses and are dependent on them for the vector transmission , movement, and replication . Trans-replication is also reported in New World begomoviruses (Nawaz-ul-Rehman et al. 2012). Betasatellites have a very conserved genome with adenine-rich region (A-rich), which is known as the satellite-conserved region (SCR). This conserved region is also present as single open reading frame (ORF) in the complementary strand in Tomato leaf curl virus-sat (ToLCV-sat) and codes for the βC1 protein (Briddon et al. 2003, 2008). The satellite-conserved region is similar to the origin of replication of geminiviruses and nanoviruses, and it has a hairpin structure with a loop sequence TAA/GTATTAC (Briddon et al. 2003). Betasatellites help in augmentation of the accumulation of their helper begomoviruses and also help in the enhancement of the symptoms in some host plants (Briddon et al. 2001; Nawaz-ul-Rehman and Fauquet 2009; Patil and Fauquet 2010; Saunders et al. 2000), which is due to βC1 protein and its role as suppressor of silencing (Cui et al. 2005). Alphasatellites and betasatellites need helper virus for replication and symptom attenuation (Idris et al. 2011).

Beside betasatellites, some begomoviruses are also associated with an additional single-stranded DNA component, which was previously called as DNA-1, and now it is known as alphasatellite (Briddon et al. 2004). These molecules are mostly half the size of begomovirus DNA components (~1375 nt) and show a common organization which consist of a single ORF and it codes for a Rep protein which shows resemblance to nanoviruses (Mansoor et al. 1999; Saunders and Stanley 1999). For insect transmission and movement within the plants, these molecules need a helper begomovirus but in host plants they are capable of self-replication (Saunders and Stanley 1999; Saunders et al. 2000). In some cases these alphasatellites have also been shown to reduce the begomovirus-betasatellite symptoms by reducing betasatellite DNA accumulation (Idris et al. 2011). Two different alphasatellites have been found to be associated with New World begomoviruses. In Brazil, they were found to associated with two bipartite begomoviruses which infect weeds (Euphorbia mosaic virus and Cleome leaf crumple virus), and in both the cases, they contain the typical conserved genome features of alphasatellites, including a gene encoding a Rep protein, an A-rich region, and a hairpin structure similar to those of alphasatellites reported from Africa (Paprota et al. 2010). The alphasatellite-like molecule reported from Venezuela was associated with the Melon chlorotic mosaic virus which is a bipartite begomovirus, and it had all the genome features of this type of DNA satellite, and its sequence is different from that of Old World alphasatellites (Romay et al. 2010).

Alphasatellites were also isolated from dragonflies which transmit mastreviruses from agricultural fields in Puerto Rico (Rosario et al. 2013). Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA) were detected in different field samples of wheat which were infected with Wheat Dwarf India Virus (WDIV) (Mastrevirus) (Kumar et al. 2014).

9.2 Betasatellites

Satellite virus and satellite RNA are commonly found associated with RNA viruses. They are defined as virus or nucleic acid that depends on a helper virus for replication , but lacks nucleotide sequence homology to the helper viral genome. In addition, satellite RNA depends on helper virus for transmission by encapsidation within the same coat protein coded by helper virus along with helper virus nucleic acid. A majority of satellites interfere with the replication of helper virus and cause attenuation in symptoms but some contribute to increased severity of symptoms (Murant and Mayo 1982; Collmer and Howell 1992; Dry et al. 1997 and Mansoor et al. 2003). No DNA satellite was found to be associated with any plant DNA viruses till 1997. However, in a prokaryotic system, bacteriophage P4 has been classified as a satellite DNA with the genome size of 11.6 kb containing 13 functional genes; the p4 DNA maintains itself as a multiple copy plasmid in the infected host cells, and it depends on helper phage for lyses (Dry et al. 1997). Unlike RNA satellite the P4 DNA bacterial phage satellite does not depend on a helper for replication and transmission. In 1997, a novel subviral agent, satellite DNA of 682 nucleotides in length, was found associated with a monopartite Tomato leaf curl virus originating from Australia (ToLCV-sat). The satellite DNA depends on the helper virus for its replication, encapsidation, and transmission and has no role in disease pathogenesis (Dry et al. 1993).

The first full-length betasatellite (1347 nts in length) was identified in 1999 in an Ageratum yellow vein virus (AYVV)-infected Ageratum conyzoides plant showing yellow vein symptoms (Saunders et al. 2000). Since then, many begomovirus-betasatellite disease complexes have been shown to be responsible for economically important diseases in different plant species in Africa and Asia, especially in China and the Indian subcontinent. These virus complexes elicit various disease symptoms, including leaf curling, enations, and yellow veins, and are major threats to crops (Mansoor et al. 2003, 2006). The complexes also cause diseases in a wide range of dicotyledonous host species within at least 37 different genera in 17 families that include vegetable crops, fiber and ornamental plants, and many weeds (Zhou 2013).

The betasatellite molecule can be defined as symptom modulating, circular, single-stranded DNA that lacks sequence homology with helper virus except the loop sequence of nonanucleotide TAATATT/AC, seen in begomoviruses, and depends on helper virus for replication and transmission (Saunders et al. 2000; Mansoor et al. 2003; Briddon et al. 2003 and Zhou et al. 2003). Most of the characterized begomoviruses associated with betasatellites are monopartite and occur in the Old World. Betasatellites are indispensable for induction of typical virus symptoms in host plants.

9.2.1 Structural Features of Betasatellite

Analysis of nucleotide sequence of betasatellite molecular data suggests that there is no obvious sequence homology with helper begomovirus DNA-A components except for the nonanucleotide sequence, TAATATT/AC. It is contrasting to what is seen in bipartite begomoviruses, wherein approximately 200 nucleotides are common for the DNA-A and DNA-B belonging to the same species. The betasatellite possesses three major regions: satellite-conserved region (SCR), adenine-rich region (a rich region), and an open reading frame βC1 (ORF βC1) (Briddon et al. 2003 and Zhou et al. 2003; Sivalingam et al. 2010). SCR is -200nt in length and consists of stem and loop (S-L) region, and it is conserved for all betasatellite molecules. The most conserved region (93–100 %) within SCR is approximately 115 nucleotides at the 5′ region of SCR (Zhou et al. 2003). The SCR may be having iteron sequence for protein (Rep), binding of which will mediate replication. This specific Rep binding sequence is not yet identified in betasatellite as in the case of bipartite DNA-A and DNA-B. However, in few betasatellite iteron sequences have been identified. In Ageratum yellow vein disease (AYVD), Saunders et al. in 2000 found that DNA-A of Ageratum yellow vein virus AYVV has GGTACTCA as its iteron, the same sequences were not found in betasatellite. But similar sequences GCTACGCA and GGTACAACA were identified in upstream of S-L portion suggesting its function as an iteron-binding sequence for Rep (Saunders et al. 2000), but experimental evidence are lacking mutation experiments, and sequence comparison between ToLCV and ToLCV-Sat in Australia revealed another stem and loop segment called S-L II (Dry et al. 1997). Based on nucleotide sequence analysis, Briddon and group (2003) found that SCR has cryptic Rep binding site for trans-replication of betasatellite by helper begomovirus and identified five-base-pair core sequences, GGN1N2N3, and a variable number of addition of nucleotide which are species specific. These iteron sequences are possibly generic recognition sequences for rep rather than species specific. Multiple alignment of SCR of betasatellite associated with Bhendi yellow vein mosaic disease (BYVMD), Cotton leaf curl disease (CLCuD), AYVD, and ToLCV-sat showed that there are no iteron sequences as presented in their helper virus , but the sequence GCTACGC occurred twice in upstream of S-L; this sequence may be an iteron sequence for rep (Jose and Usha 2003). The Rep binding site for satellite DNAs associated with Cotton leaf curl Gezira virus (CLCuGV) originating from the Nile Basin consists of directly repeated sequence (CGGTACTCA) and an inverted repeated sequence (TGATGACCG) occurring in the text of 17 nucleotide motif (Idris et al. 2005).

A-rich region is approximately 160–280 nucleotides in length, located upstream of SCR, from 750 to 1000th nucleotide coordinate (Zhou et al. 2003). This region is maintained in all the betasatellite molecules including ToLCV-sat from Australia. It is hypothesized that A-rich region may arise due to duplication of the sequence that they may act as “stuffer” (a region of arbitrary sequences) required to the size of the betasatellite molecule to get encapsidated inside the coat protein (Saunders et al. 2000). However, Briddon et al. (2003) suggest it to have a function in complementary strand DNA replication .

The betasatellite encodes many ORFs with the predicted protein molecular weight of more than 4kDa, but only one ORF is present in complementary strand (approximately from 550 to 200th nucleotide coordinate), encode−13.5 kDA to−17.5 kDa protein called βC1, which is known to be functional. ORF βC1 is positionally conserved in all betasatellites characterized so far, from different geographical locations and diverse host species. There is a TATA box upstream of the ORF and polyadenylation signal for transcription to occur (Briddon et al. 2003; Mansoor et al. 2003; Bull et al. 2004 and Cui et al. 2004a, b).

In a large number of infected samples analyzed, defective deletion mutants of betasatellite have been found to occur in nature. The mutant maintains SCR and A-rich region but the deletion was observed in ORF βC1 (Briddon et al. 2003 and Bull et al. 2004).

9.2.2 Betasatellites Associated with Begomovirus

Betasatellite molecules isolated and characterized so far are found to be associated with many diseases caused by monopartite begomoviruses (Briddon et al. 2001, 2003; Mansoor et al. 2003; Saunders et al. 2000; Zhou et al. 2003, Radhakrishnan et al. 2004, Jose and Usha 2003; Bull et al. 2004; Xiong et al. 2005; Singh et al. 2011, 2012) and some are found associated with bipartite begomoviruses (Malathi et al. 2004; Rouhibakhsh and Malathi 2005). As far as identification of betasatellite is concerned; a major breakthrough came while investigating Ageratum yellow vein disease (AYVd), which was known to be caused by monopartite begomovirus having DNA-A alone. Inoculation with DNA-A alone did not produce any typical symptoms in ageratum as seen in the field (Stanley et al. 1997). Rigorous attempts made to isolate another component, DNA-B were unsuccessful. Several recombinant DNA of approximately half the size to helper genome were isolated from an yellow vein disease infected Ageratum conyzoides plants and they were characterized.

Some of the recombinant clones had part of DNA-A sequence and sequence of unknown origin. Using primers in PCR approach betasatellite and alphasatellite were isolated and sequenced (Mansoor et al. 1999 and Saunders et al. 2000). Sequenced data revealed that the recombinant/defective components had the sequence of unknown origin and shared no homology with helper DNA-A except for TAATATTAC.

Betasatellites are capable of being trans-replicated by different begomoviruses, so a variety of begomovirus-betasatellite complexes can occur. For example, the cotton leaf curl disease (CLCuD) in Pakistan is reportedly caused by association of a single betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB), with at least six begomovirus species, either as single or multiple infections (Nawaz-ul-Rehman et al. 2012). African CLCuD-associated begomovirus Cotton leaf curl Gezira virus (CLCuGV) was identified in cotton from southern Pakistan where CLCuMuB is known to be found (Tahir et al. 2011). It has also been reported that true monopartite begomoviruses, such as Tomato yellow leaf curl virus (TYLCV) and Papaya leaf curl China virus (PLCCNV), can trans-replicate betasatellites (Zhang et al. 2009; Zhang et al. 2010). These observations raise an alarming scenario in which true monopartite begomoviruses may form new disease complexes by acquiring other begomovirus betasatellites in mix-infected plants. Indeed, divergent isolates of Tomato yellow leaf curl disease (TYLCD) from Oman is associated with a betasatellite (Khan et al. 2008), and a severe symptom phenotype in tomato in Mali is caused by a novel begomovirus-betasatellite complex resulting from reassortment (Chen et al. 2009). Cotton leaf curl Gezira betasatellite (CLCuGB), initially identified in the Nile basin, has been identified in West Africa, associated with diseased okra and tomato (Chen et al. 2009; Kon et al. 2009; Shih et al. 2009; Tiendrebeogo et al. 2010; Idris et al. 2014).

9.2.3 Diversity of Betasatellite

Information on the existence and diversity of DNA satellite molecules associated with monopartite begomoviruses has been mainly from Asia (Bull et al. 2004; Nawaz-ul-Rehman and Fauquet 2009; Sivalingam et al. 2010). Identification of betasatellite molecules, which are associated with monopartite begomoviruses widely distributed in the Old World, led to investigation on the molecular variability in the betasatellite. Considerable variation has been found in nucleotide sequence of full-length betasatellite. A-rich region and amino acid sequence of ORF βC1. However, conservation was also found in the ORF βC1. A major initiative was taken to determine the variability in Yunana province of China (Zhou et al. 2003), Pakistan, India, Egypt, Singapore, UK, (Briddon et al. 2003) and East and South east Asian countries (Bull et al. 2004). The nucleotide sequence of complete betasatellite was compared with betasatellite earlier reported from CLCuD, Bhendi yellow vein mosaic disease (BYVMD) and Agretum yellow vein disease (AYVD) originating from India, Pakistan and Singapore (Saunders et al. 2000; Jose and Usha 2003, Radhakrisnan 2003, Zhou et al. 2003). Analysis of nucleotide sequence of eighteen betasatellite molecules with their corresponding helper DNA-A was done. This analysis include 13 betasatellite associated with Tomato yellow leaf curl China virus (TYLCCNV), four betasatellite with Tobacco curly shoot virus (TbCSV) and one with Cotton leaf curl Multan virus (CLCuMV). Based on their analysis, they proposed a species concept that betasatellite molecules sharing 72–99 % identity belong to one species and betasatellite sharing 36–57 % identity to be considered as different species, with the exception of betasatellite associated with Malvastrum Yellow Vein Virus (MYVV-Y47) (62–67 % identity) and betasatellite with Cotton leaf curl Rajasthan virus CLCuRaV .

Briddon et al. 2003 showed that nucleotide sequence similarity was 49–99 % between the betasatellite molecules , but for betasatellite molecules originating from different disease and/or geographical locations (called unrelated betasatellite) nucleotide similarity was up to 71 % indicating their association with their helper /disease in a geographical isolation. Amino acid sequence comparison of βC1 showed low level of sequence identity (35–50 %) between βC1 of betasatellite molecules.

Bull et al. 2004 attempted to understand the diversity within betasatellite isolated from East and Southeast Asia. They showed that there was more diversity within the region due to the limited movement of begomovirus-betasatellite complex and suggest that all the betasatellite molecules originated from a common ancestor and further got adapted according to the host.

Betasatellites are reported from various crops and along with different viruses in India. This shows their diversity and emergence (George et al. 2014; Kumar et al. 2014; Srivastava et al. 2013a, b; Singh et al. 2012) . Betasatellites reported from different countries is depicted in the world map in Fig. 9.1. Betasatellites reported from various crops is being tabulated in the Table 9.1.

Fig. 9.1
figure 1

World wide distribution of betasatellites associated with begomoviruses . Red color corresponds to countries all over the world from where betasatellites have been reported. Numbers on the map are indicative of specific area in the country from where they are reported and these number also correspond to betasatellite name in the Table 9.1

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Table 9.1 List of begomovirus -associated betasatellites reported on different crops from various parts of the world
Full size table

9.2.4 Recombinant Betasatellite

Evidence for recombinant betasatellite is based on nucleotide sequence analysis. The betasatellite associated with CLCuRV can be divided into two parts (Zhou et al. 2003). Part one is 184 nt located between 1065 and 1269, which has only 62 % identity with betasatellite of CLCuMV, but rest of the region showed 98 % similarity with the same betasatellite. This indicate that betasatellite associated with CLCuRV and CLCuMV evolved by recombination (Zhou et al. 2003). Another example is betasatellite associated with Tomato leaf curl disease (TomLCD) in Pakistan (TomLCDβ01-Pak). This betasatellite molecule evolved by natural recombination between betasatellite associated with TomLCD and CLCuD (CLCD β02-Pak) as the SCR of TomLCDβ02-Pak showed 90 % nucleotide identity with TomLCDβ02-Pak and only 77 % with CLCDβ02-Pak. Another betasatellite associated with Okra leaf curl disease (OLCD) (OLCD β03-Pak) also seems to have evolved through recombination (Briddon et al. 2003).

9.2.5 Role of Betasatellite in Pathogenesis

Betasatellite has been found essential in the pathogenesis and expression of typical symptoms of enation, vein thickening, extreme leaf crinkling, twisting of petioles, etc. induced by the begomoviruses causing disease in ageratum, bhendi, cotton, etc. when betasatellite is co-inoculated with DNA- A of helper virus (Briddon et al. 2001; Mansoor et al. 2003; Zhou et al. 2003; Jose and Usha 2003).

In bipartite begomoviruses DNA-A and DNA-B have specific iteron sequence in a species-specific manner. This assumes replication of cognate DNA-B by the Rep encoded by corresponding DNA-A. Similarly, betasatellite associated with different diseases like Agretum yellow vein disease (AYVD), Agretum yellow leaf curl diseases (AYLCD), Cotton leaf curl diseases (CLCuD), Honeysuckle yellow vein mosaic disease (HYVMD), Okra leaf curl disease (OLCD), Okra yellow vein mosaic disease (OYVMD), Tobacco leaf curl disease (TbLCD) and Tomato leaf curl disease (ToLCD) have been shown to be replicated by Rep protein encoded by DNA-A of different begomovirus species (Briddon et al. 2003; Bull et al. 2004). It indicates that the betasatellite has relaxed specificity for Rep to get replicated. Inoculation of DNA-A of Sri Lankan cassava mosaic virus (SLCMV) with betasatellite associated with AYVD produced typical yellow vein symptoms in Ageratum conyzoids, but symptoms develops when DNA-A alone is inoculated. These results indicate biological activity and requirement of betasatellite in pathogenesis and its relaxed specificity for Rep encoded by SLCMV (Saunders et al. 2002a, b). The betasatellite associated with CLCuD in Pakistan is also shown to replicate with the help of DNA- A of monopartite begomoviruses like CLCuMV, Cotton leaf curl Kokhran virus (CLCuKV), Cotton leaf curl Alabad virus (CLCuAV) and Papaya leaf curl virus (PaLCV) (Mansoor et al. 2003). ToLCV –sat from Australia has been shown to replicate not only by other begomovirus species but also by other genus; Curtovirus (Beet curly top virus) (Dry et al. 1997). So the specificity for Rep to replicate TLCV-sat DNA appears to be more relaxed. Mutation experiments have shown the role of ORF βC1 in the pathogenesis of TYLCCNV-y10 DNA-A; DNA-A when co-inoculated with betasatellite having in-frame AUG mutated ORF βC1 produced milder symptoms, which were comparable with DNA-A alone inoculated plants; however the replication of betasatellite plays an important role in symptom development but which is not essential for replication of betasatellite (Zhou et al. 2003 and Cui et al. 2004a, b).

Two major phenotypic symptoms are observed in disease complex with betasatellite like the vein yellowing as found in Ageratum yellow vein and leaf curling, vein swelling, vein darkening, ectopic enations, etc. as in CLCuD. Vein darkening is caused by replacement of spongy parenchyma by palisade parenchyma. Mansoor et al. 2003 have suggested that abnormal cambium activity in phloem parenchyma leads to formation of secondary vascular elements leading to downward leaf curling.

The role of βC1 gene of betasatellite in symptom development has been confirmed in transgenic N. benthamiana plants having βC1 of betasatellite associated with AYVD. These plants develop malformed leafy structure; severely distorted stems and leaves, vein greening , etc. as in diseased plants (Saunders et al. 2004 and Cui et al. 2004a, b). Leaf distortion and severe curling symptoms were produced in and transgenic N. benthamiana and N. tabacum βC1 gene associated with TYLCCNV-Y10.

Betasatellite has also been shown to contribute to expansion of host range of the associated begomoviruses. SLCMV, a bipartite begomovirus does not infect ageratum, but, when SLCMV DNA-A is co- inoculated with betasatellite of AYVD in ageratum, is infected producing typical yellow vein symptoms (Saunders et al. 2002a, b). The role of betasatellite in host range determination has also been demonstrated for TYLCCNV-Y10. Co-inoculation of TYLLCNV-Y10 DNA-A with associated betasatellite having deletion, infection was obtained in N. benthamiana and N. glutinosa but not in N. tabacum and Lycopersicum esculentum plants (Qian and Zhou 2005).

Co-inoculation of begomoviruses with the associated betasatellite produces more severe symptoms than in inoculation with the respective begomovirus alone. The co-inoculated plants also have high level of accumulation of helper begomovirus DNA-A. Earlier the enhanced severity in disease symptoms was considered to be due to the infection of betasatellite on DNA-A replication (Saunders et al. 2000). But the recent evidences show that βC1 protein of betasatellite of TYLCCNV-Y10 could interact with host factor(s) to induce symptoms. They either act as suppressor of gene silencing or interfere with host defense system thereby allowing more efficient systemic infection of the plants (Cui et al. 2004a, b, 2005; Saunders et al. 2004) demonstrated that βC1 protein of DNA-β of TYLCCNV-Y10 and TbCSV-Y35 could bind both single-stranded and double-stranded DNA in size and sequence nonspecific manner. They could find that βC1 protein accumulation is the key requirements for symptom induction and silencing suppression.

Infectivity assays of Tomato leaf curl New Delhi virus (ToLCNDV) and Chili leaf curl betasatellite (ChLCB) were conducted by Akhtar et al. 2014, DNA-A and DNA-B of ToLCNDV isolated from chilies and tomato were found to be infectious and produced leaf curl symptoms when inoculated on Nicotiana benthamiana by biolistic gun method. Co-inoculation of ToLCNDV with ChLCB resulted in the severity of disease symptoms.

Association of betasatellites with bipartite begomoviruses is rare and has only been reported in India, where the role of a betasatellite in pathogenesis of a bipartite begomovirus, Tomato leaf curl New Delhi virus (ToLCNDV), was investigated (Sivalingam and Varma 2012). Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A alone could infect tomato and Nicotiana benthamiana and induces mild symptoms. When these two hosts were co-inoculated with ToLCNDV DNA-A and ToLCNDV DNA-B or ToLCNDV DNA-A and CLCuMuB, typical leaf curling symptoms developed, but co-infections with all three components resulted in much more severe disease symptoms.

In addition, ToLCNDV DNA-A and DNA-B accumulated to six to eightfold higher levels in plants co-inoculated with all three components than in plants co-inoculated with only DNA-A and DNA-B. Like many other viral pathogenicity determinants, the βC1proteins can function as RNA silencing suppressors. The reported βC1 suppressors include TYLCCNB-βC1, CLCuMuB-βC1, and βC1 proteins of betasatellite associated with Bhendi yellow vein mosaic virus (BYVMV), Tomato leaf curl Java virus (ToLCJAV), and Tomato leaf curl China virus (ToLCCNV) (Cui et al. 2005; Gopel et al. 2007; Sharma et al. 2011). Unlike most geminiviruses studied, Tomato yellow leaf curl China virus (TYLCCNV) is susceptible to cytosine methylation and is not effective in suppressing TGS of a green fluorescent protein transgene in plants. In contrast, βC1 from TYLCCNB is able to mediate TGS suppression (Sunter et al. 1994).

Betasatellites enhance the accumulation of their helper begomoviruses by increasing the symptoms induced in some host plants (Briddon et al. 2001; Nawaz-ul-Rehman and Fauquet 2009; Patil and Fauquet 2010; Saunders et al. 2000), it is most probably due to the βC1 protein gene’s silencing suppressor activity (Cui et al. 2005; Saeed et al. 2005).

9.2.6 Evolutionary Relationship

Origin of betasatellite is still unknown; however, certain speculations have been made based on available information. Bipartite begomoviruses might have evolved from an ancestral monopartite virus by component capture and duplication along with acquired novel genetic material. DNA-A donated its origin of replication to DNA-B while evolving and acquired additional genetic material. Alternatively, the monopartite virus donated origin of replication to betasatellite originating from an unknown progenitor and adopted along with DNA-A by component capture mechanism; component capturing led to extension of the host range, adaptation in the new environmental condition producing novel disease. Variability arose by recombination mechanism which provided enormous scope for diversification and modification of biological properties to allow adaptation to new ecological niches (Mansoor et al. 2003). Recombination, a powerful tool for evolution of betasatellite has been shown in several betasatellite associated with AYVD and CLCuD by different research groups (Zhou et al. 2003; Briddon et al. 2003).

9.2.7 Betasatellite Used as Vector

ßC1 gene of betasatellite associated with TYLCCNV-Y10 isolate was replaced with multiple cloning site (MCS) facilitating insertion of gene such as proliferating cell nuclear antigen (PCNA), phytoene desaturase (PDS) and sulfur (Su) gene or green fluorescent protein (GFP) in the MCS of betasatellite. Such constructs were inoculated with TYLCCNV-Y10 DNA-A separately on N. benthamiana, N. glutinosa, N. tabacum and tomato plants. Silencing of the above mentioned gene was found in all the plants. Results showed that this was due to βC1 gene which might have acted as a suppressor of gene silencing. It opens the possibility of the use betasatellite mediated vector system in functional genomics (Tao and Zhou 2008).

9.2.8 Emerging Betasatellites

Betasatellites are reported from South Asia, East Asia, Southeast Asia, Africa, the Middle East, the UK, Australia and New Zealand. That is from the Old World; there is no report of betasatellites from the New World. A number of betasatellites reported along with host plants are given in the Table 9.1 and also illustrated in the Fig. 9.1.

9.3 Alphasatellites

In 1999 a Nanovirus-like DNA component associated with Yellow vein disease of Ageratum conyzoides was reported (Saunders and Stanley 1999) which was later called as Agretum yellow vein alphasatellite. This was the first evidence of association of satellite like particles with geminiviruses . Thereafter number of alphasatellites are being reported from all over the world from different crops, viz., okra, cassava, chili, ageratum, sunflower , cotton, croton, hollyhock, Malvastrum, tobacco, tomato, and mesta. In 1999 from Pakistan, a novel circular ssDNA associated with cotton leaf curl disease was reported (Mansoor et al. 1999).

Alphasatellite genomes are approximately1,375nts and encode a single ORF (alpha-Rep). The alpha-Rep ORF encodes a 315 amino acid protein of ∼ 37 kDa that resembles nanovirus Reps. Alpha-Rep sequences are more conserved than the full-length alphasatellite sequences (Briddon et al. 2004; Xie et al. 2010). A-rich region ∼150- to 200-nt has an A content between 46 % and 58 % and is the only feature that can be used to distinguish begomovirus alphasatellites from nanovirus Rep-encoding components. It has been suggested that the A-rich sequences may only function to increase sizes of alphasatellite molecules to half the size of the begomovirus components (Briddon et al. 2004). The predicted alphasatellite hairpin structure has a loop containing a nonanucleotide, TAGTATTAC, common to nanoviruses that is also similar to the analogous TAATATTAC nonanucleotide sequence in begomovirus loop structures.

Alphasatellites have no contribution to symptoms induced by begomovirus-betasatellite disease complexes and appear to affect betasatellite replication but do not affect helper virus replication. Some alphasatellites can attenuate disease symptoms caused by begomovirus-betasatellite complexes in the early stages of infection (Nawaz-Ul-Rehman et al. 2010).

Alphasatellite was identified in Ageratum in Singapore (referred to as DNA-2) (Saunders et al. 2002a, b). DNA-2 type alphasatellite members have been identified in Oman (Idris et al. 2011) and India (Zaffalon et al. 2012). Although all these members contain conserved alphasatellite genome features, the DNA-2 type molecules are less homogeneous and have less than 50 % nucleotide sequence identity with each other. The DNA2 type alphasatellite identified in Oman can attenuate begomovirus symptoms and reduce accumulations of betasatellites. Alphasatellites were though discovered almost 16 years ago, still very less information is available about their function(s). Begomovirus-associated defective satellites have also been identified in malvaceous plants in Cuba and whiteflies in Florida, thus indicating the natural occurrence of this type of satellite molecule (Fiallo-Olivéa et al. 2012).

9.3.1 Origin and Evolution of Alphasatellites

The alphasatellites most likely originated from nanoviruses through adaptation of a nanovirus component by becoming encapsidated in the begomovirus coat protein for whitefly transmission after vector feeding on plants co-infected with their begomovirus and nanovirus progenitors (Patil and Fauquet 2010). A related class of alphasatellites has also been found associated with viruses in the Nanoviridae family. Most alpha Reps are highly conserved, but alphasatellites found in the New World are more diverse and clarification of their evolution requires additional sequence studies of a wider range of isolates (Briddon et al. 2014).

9.3.2 Emerging Alphasatellites

Unlike betasatellites, the presence of alphasatellites, as in the case of Ageratum yellow vein Singapore alphasatellite (AYVSGA), Tobacco curly shoot alphasatellite (TbCSA), Gossypium darwinii symptomless alphasatellite (GDarSLA), and Gossypium mustelinum symptomless alphasatellite (GMusSLA) in plants infected with begomovirus-betasatellite complexes, reduces the accumulation of betasatellite (Wu and Zhou 2005; Idris et al. 2011; Nawaz-ul-Rehman et al. 2010) and viral symptoms (Wu and Zhou 2005; Idris et al. 2011). The coding region is most closely related to those of nanoviral Reps. Though alphasatellites can replicate autonomously, but they require helper virus for insect transmission and systemic spread in plants (Saunders et al. 2000; 2002a, b; Saunders and Stanley 1999). Alphasatellites are being reported from all over the world from number of crops. In mixed infections where both alphasatellite and betasatellite particles were used with helper begomovirus, alphasatellite modulated the begomovirus-betasatellite pathogenicity by interfering with βC1 a key virulence factor (Idris et al. 2011).

Occurrence of alphasatellites in different countries is given in Fig. 9.2 and detailed list of alphasatellites and crops on which they are reported is mentioned in Table 9.2.

Fig. 9.2
figure 2

World wide distribution of alphasatellites associated with begomoviruses. Red color corresponds to countries all over the world from where alphasatellites have been reported. Numbers on the map are indicative of specific area in the country from where they are reported and these number also correspond to alphasatellite name in the Table 9.2

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Table 9.2 List of begomovirus -associated alphasatellites reported on different plants from various parts of the world
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9.3.3 Role of Alphasatellites

Alphasatellites were first detected when they were found associated with disease complexes of DNA β and helper virus (Briddon and Stanley 2006; Mansoor et al. 2003; Stanley 2004). It is speculated that after co-infection of begomoviruses and nanoviruses to host plants which are common host of both viruses these might have adapted to whitefly transmission (Mansoor et al. 1999; Saunders et al. 2002a, b; Saunders and Stanley 1999).

Alphasatellites can replicate autonomously, but a helper virus is required for insect transmission and for systemic spread in plants. (Saunders et al. 2000; 2002a, b; Saunders and Stanley 1999). In case of alphasatellites no effect has been reported on the development of symptoms (Briddon et al. 2004). In some reports an unusual class of alphasatellites has been shown to attenuate begomovirus-betasatellite symptoms by reducing betasatellite DNA accumulation (Idris et al. 2011). The alpha-Rep proteins encoded by two nonpathogenic alphasatellites: Gossypium darwinii symptomless alphasatellite, Gossypium mustelinium symptomless alphasatellite (GDarSLA and GMusSLA) associated with Cotton leaf curl Rajasthan virus (CLCuRaV) can interact with CLCuRaV Rep proteins (Nawaz-Ul-Rehman et al. 2010).

Alphasatellites possess a gene encoding a protein most closely related to the replication initiator (Rep) protein of nanoviruses. Consequently, they are capable of autonomous replication in plant host cells but require the helper begomovirus for movement within the plant and for insect transmission (Briddon et al. 2004). Alphasatellite molecules have been shown to ameliorate symptoms (Idris et al. 2011; Nawaz-ul-Rehman et al. 2012; Wu and Zhou 2005). Their presence has been reported not only in cultivated plants (tomato, cotton, okra, tobacco, watermelon), but also in ornamental and wild plants (Althea rosea, Ageratum conyzoides, Cleome affinis, Euphorbia spp., Hibiscus rosa-sinensis) infected by monopartite (Mubin et al. 2009; Idris et al. 2011; Tiendrebeogo et al. 2010) and bipartite (Paprotka et al. 2010, Romay et al. 2010) begomoviruses. Saunders and Stanley (Idris et al. 2011) first reported that alphasatellites can systemically infect Nicotiana benthamiana in the presence of cassava mosaic geminiviruses (CMGs) , particularly African cassava mosaic virus.

9.4 Conclusion

Over the past 15–16 years, associations of betasatellites with begomoviruses have emerged as serious threats to a wide range of crops in the whole world. Betasatellites can be trans-replicated by several different begomoviruses, and trans-replication by true monopartite begomoviruses has the potential to form new disease complexes through acquisition of various begomovirus betasatellites from mixed infected plants. Moreover, betasatellites play important roles in determining begomovirus host ranges and hence could lead to emergence of new complexes that can cause severe crop epidemics .

Alphasatellites were first identified in association with monopartite begomovirus infections in the Old World that are known to harbor betasatellites and more recently in plants infected with bipartite begomoviruses. The biological functions of alphasatellites are still obscure. These satellites have emerged due to several reasons most important being the movement of infected material and vectors due to poor quarantine facilities. Also vector Bemisia tabaci has also extended its host range and thus transmitting virus to wide range of crops.

Monopartite begomovirus encode all the genes which are required for a successful infection. Most of these genes code for multifunctional proteins which adds further complexity in their interaction with host proteins, and their de novo creation. This shows the ability of begomoviruses and their associated satellites to rapidly evolve in response to selection pressures such as host plant resistance .