Skip to main content

Roles of two distinct alphasatellites modulating geminivirus pathogenesis



Alphasatellites are small coding DNA satellites frequently associated with a begomovirus/betasatellite complex, where they are known to modulate virulence and symptom development. Two distinct alphasatellites, namely, Cotton leaf curl Multan alphasatellite (CLCuMuA), and Gossypium darwinii symptomless alphasatellite (GDarSLA) associated with Cotton leaf curl Multan virus-India (CLCuMuV-IN) and Ludwigia leaf distortion betasatellite (LuLDB) were found to be associated with yellow mosaic disease of hollyhock (Alcea rosea) plants. In this study, we show that alphasatellites CLCuMuA and GDarSLA attenuate and delay symptom development in Nicotiana benthamiana. The presence of either alphasatellites reduce the accumulation of the helper virus CLCuMuV-IN. However, the levels of the associated betasatellite, LuLDB, remains unchanged. These results suggest that the alphasatellites could contribute to the host defence and understanding their role in disease development is important for developing resistance strategies.


Tandem repeat constructs of two distinct alphasatellites, namely, CLCuMuA and GDarSLA associated with CLCuMuV-IN and LuLDB were generated. N. benthamiana plants were co-agroinoculated with CLCuMuV and its associated alphasatellites and betasatellite molecules and samples were collected at 7, 14 and 21 days post inoculation (dpi). The viral DNA molecules were quantified in N. benthamiana plants by qPCR. The sequences were analysed using the MEGA-X tool, and a phylogenetic tree was generated. Genetic diversity among the CLCuMuA and GDarSLA was analysed using the DnaSP tool.


We observed a reduction in symptom severity and accumulation of helper virus in the presence of two alphasatellites isolated from naturally infected hollyhock plants. However, no reduction in the accumulation of betasatellite was observed. The phylogenetic and genetic variability study revealed the evolutionary dynamics of these distinct alphasatellites , which could explain the role of hollyhock-associated alphasatellites in plants.


This study provides evidence that alphasatellites have a role in symptom modulation and suppress helper virus replication without any discernible effect on the replication of the associated betasatellite.


Viruses of the genus Begomovirus, family Geminiviridae have a small circular ssDNA genome and are transmitted by whiteflies [1]. The genome of begomoviruses are bipartite (components known as DNA A and DNA B) or monopartite (a single component similar to DNA A of abipartite begomovirus) [1]. At least three classes of circular ssDNA satellites associated with begomoviruses have been described: betasatellites [2], alphasatellites [3] and deltasatellites [4, 5].

Betasatellites (1.5–1.7 kb) genome contain a satellite conserved region (SCR) and a single gene, βC1. Betasatellites depend on the helper begomovirus for their replication, movement and transmission. βC1 is known to be a symptom determinant and thus, enhances the symptoms in some pathosystems [6,7,8]. It is also known to suppress transcriptional (TGS) and post-transcriptional gene silencing (PTGS) [7, 9, 10].

Alphasatellites (1.5–1.7 kb) associate with the begomovirus/betasatellite complex and depend on the helper begomovirus only for their movement in plants and vector transmission. They encode their own nanovirus-like replication initiator protein (Alpha-Rep) which enables them to replicate autonomously in plant cells [11, 12]. The Rep of some alphasatellites is known to suppress PTGS and TGS [13, 14]. Alphasatellites have a role in pathogenicity as well. At least two alphasatellites have been shown to attenuate disease symptoms and reduce the accumulation of the helper begomovirus and its associated betasatellite [15, 16]. In contrast, the association of alphasatellites with Wheat dwarf India virus (WDIV), a mastrevirus, increases WDIV accumulation and reduces viral small interfering RNA (VsiRNA) levels [17].

Previously, we have reported the presence of an isolate of monopartite Cotton leaf curl Multan virus-India (CLCuMuV-IN, Accession no. MG373551) and Ludwigia leaf distortion betasatellite (LuLDB, Accession no. MG373553) from hollyhock (Alcea rosea) plants exhibiting yellow mosaic symptoms [18]. Further, we isolated two alphasatellites Cotton leaf curl Multan alphasatellite (CLCuMuA, Accession no. MG373558) and Gossypium darwinii symptomless alphasatellite (GDarSLA, Accession no. MG373559) from the same hollyhock plants [18]. In this study, we demonstrate the role of CLCuMuA and GDarSLA in pathogenicity and more specifically their ability to delay and attenuate symptom severity and reduce the accumulation of the helper virus CLCuMuV-IN, with no apparent effect on the accumulation of the associated betasatellite LuLDB.


Construction of agroinfectious clones

Hollyhock plants exhibiting typical leaf curling symptoms, veinal chlorosis, vein thickening, and yellow mosaic symptoms (Fig. 1) were collected and various geminivirus DNAs were previously cloned from these samples [18]. These clones available in our laboratory were used for generation of infectious clones. Partial tandem repeat constructs (PTR) of Cotton leaf curl Multan virus-India (CLCuMuV-IN, Accession no. MG373551) and Ludwigia leaf distortion betasatellite (LuLDB, Accession no. MG373553) and two alphasatellites Cotton leaf curl Multan alphasatellite (CLCuMuA, Accession no. MG373558) and Gossypium darwinii symptomless alphasatellite (GDarSLA, Accession no. MG373559) were generated in this study. For CLCuMuV-IN, the EcoRV (300 nt)-XhoI (2077 nt) fragment containing the CR region was cloned into the pBluescript II KS (+) vector, followed by the monomeric DNA-A molecules to generate the PTR construct. Similarly, for CLCuMuA alphasatellite, approximately 1.2 kb EcoRI (515 nt)-BamHI (1526 nt) fragment and for GDarSLA, EcoRI (508 nt)-SalI (1355 nt) fragment of the satellite DNA was digested and cloned into the pBluescript II KS (+) vector and further mobilized into the binary vector pCAMBIA2300 at XbaI and KpnI site. For LuLDB, the 500 bp of KpnI (507 nt)-SalI (1307 nt) fragment was cloned in pCAMBIA2300 followed by ligation of the full-length KpnI digested monomer fragment to generate the PTR constructs of betasatellite [19, 20]. Further, these infectious clones were transformed into the Agrobacterium EHA105 for infectivity analysis in N. benthamiana plants.

Fig. 1
figure 1

Symptoms induced by the begomoviruses on hollyhock (A. rosea) plants showing a leaf curling and b yellowing of leaves inthe field condition. Non-symptomatic or healthy plants were also shown adjacent to the diseased one for the identification of the diseased plants

Agrobacterium-mediated inoculation and infectivity analysis

N. benthamiana wild-type plants were grown in an insect-free growth chamber at 25 ± 2 °C, 60–70% relative humidity, and 16/8 h (light/dark) photoperiod. Agrobacterium tumefaciencs strain EHA105 cultures harbouring agroinfectious clones of CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA were grown overnight at 28 °C in Luria–Bertani broth supplemented with 50 µgml−1 kanamycin. Agrobacterium cultures were mixed in equal ratio  and N. benthamiana plants were inoculated at the four leaves stage as described by [21]. The inoculated plants were monitored for the appearance of symptoms under insect-proof controlled conditions for 30 days. A. tumefaciencs starin EHA105 harbouring the vector pCAMBIA2300 was used for mock inoculation. The agroinoculated plants were regularly scored for symptoms on the scale of 0 to 5 for 30dpi [22]. Symptom severity scale, 0-no symptoms, 1-very mild yellowing, 2-mild yellowing with downward leaf curling, 3-severe yellowing of leaves with downward leaf curling and vein thickening, 4-severe yellowing of leaves with downward leaf curling, vein thickening and stunted plant growth, 5-severe yellowing of leaves with downward leaf curling, vein thickening and stunted plant growth leading to plant death. The uppermost leaves of inoculated N. benthamiana plants were collected at 7, 14 and 21 dpi. The presence of CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA was checked by PCR. The infectivity analysis was repeated a minimum of three times.

Phylogenetic relationship of alphasatellite and alpha-rep protein

Nucleotide sequences of 43 full-length alphasatellites species were retrieved from the NCBI GenBank (Additional file 1: Table S1) and their phylogenetic relatedness with CLCuMuA and GDarSLA was generated using the MEGA-X tool [23, 24]. Both CLCuMuA and GDarSLA belongs to the largest genus Colecusatellite of the family Alphasatellitidae and have a single protein-encoding region α-Rep of 315 amino acids in length. To further decipher the evolutionary pattern of α-Rep protein of CLCuMuA and GDarSLA (19 isolates), a phylogenetic dendrogram was generated by the MEGA-X tool by using maximum-likelihood statistical method, Jonson-Tylor-Thornton (JTT) amino acid substitution model and 1000 bootstrap replicates.

Genetic variability analysis of alphasatellites

To understand the genetic variability among the cognate (CLCuMuA) and non-cognate (GDarSLA) alphasatellite, 94 full-length nucleotide sequences for GDarSLA and 169 full-length nucleotide sequences for CLCuMuA were selected from the NCBI GenBank (Additional file 1: Table S2). The parameter used for the analysis was; the total number of segregating sites (s), the total number of mutations (η), nucleotide diversity (π), average number of nucleotide differences between sequences (k), estimation of the population mutation rate based on the total number of s-value (θ–w), estimation of the population mutation rate based on the total number of mutations (θ–η), and gene diversity (Hd). These parameters were analysed by the DNA sequence polymorphism tool (DnaSP V 6.12.03) [25, 26].

Quantitative detection of viral DNAs by real time-PCR (qPCR)

Primers were designed for CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA using Primer Quest™ tool of Integrated DNA Technologies, Inc. USA (IDT) online software (Additional file 1: Table S3). qPCR reaction was set up using total genomic DNA isolated from infected N. benthamiana leaves and SYBR green reaction chemistry. For preparing a standard curve, restriction digestion linearized full-length clone of CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA were serially diluted ten folds to get a range of 1010–102 viral genomic copies per 2 µl plasmid. Plasmid copy number per microliter was calculated and the plasmid dilutions thus obtained was subjected to real-time PCR (Additional file 1: Fig. S1) [27]. Cycling parameters used were as follows: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 3 min (DNA polymerase activation), and 40 cycles, each consisting of 30 s at 95 °C (denaturation) and 20 s at 60 °C (annealing) and 20 s at 72 °C (extension). Dissociation analysis was performed by incubating the reaction at 95 °C for 15 s, annealing at 60 °C for 20 s and 95 °C for 20 min. For each time point and inoculation combination, six samples each with three technical replicates were tested [28, 29]. Statistical significance was calculated using ANOVA and the graphs were made using GraphPad Prism 9 (


Sequence and phylogenetic analysis of alphasatellite

Sequence comparison of the two alphasatellites revealed that CLCuMuA (size 1571 bp) and GDarSLA (size 1377 bp) shared 87.53% nucleotide sequence identity. However, the amino acid length of both the α-Rep of satellites was the same (315 aa). The phylogenetic analysis grouped CLCuMuA [India:New Delhi: Hollyhock: 16] with CLCuMuA-PK [Pakistan: Cotton: 99], while GDarSLA [India:New Delhi: Hollyhock: 16] with GDarSLA [Pakistan: Gossypium davidsonii: 09]. The two alphasatellites appear to diverge from a close common ancestor (Fig. 2). Alphasatellites encode a single protein, replication initiator protein (α-Rep) and are capable of self-replication. The CLCuMuA are found to be associated with several families of plants such as Malvaceae (Alcea rosea, Gossypium hirsutum, Abelmoschus esculentus), Poaceae (Triticum aestivum), Cucurbitaceae (Luffa aegyptiaca), Solanaceae (Solanum nigrum), and Conolvulaceae (Ipomoea batatas) [30,31,32]. Whereas, GDarSLA has been  majorly reported from the Malvaceae family (Abelmoschus esculentus, Gossypium hirsutum, Alcea rosea, Gossypium davidsonii, and Gossypium mustelinum) of plants (Fig. 3) [25, 33]. Evolutionary divergence of cognate alphasatellite α-Rep protein suggests, this alphasatellite infects several families of crop plants. However, non-cognate alphasatellite also infect some non-cultivated host plants such as G. mustelinum and G. davidsonii apart from the cultivated crop (A. esculentus) plants [34].

Fig. 2
figure 2

Phylogenetic relationships of Cotton leaf curl Multan alphasatellite (CLCuMuA, Accession No. MG373558) and Gossypium darwinii symptomless alphasatellite (GDarSLA, Accession No. MG373559) cloned from symptomatic hollyhock plants. Begomovirus associated alphasatellites included in this analysis were selected from NCBIBLAST results and the phylogenetic tree was constructed using the MEGA-X tool. In each case, the database accession number is given. The number at major mode indicates the percentage bootstrap confidence score for 1000 replicates. The distance scale bar denotes the rate of nucleotide substitution per site

Fig. 3
figure 3

Phylogenetic relatedness of Cotton leaf curl Multan alphasatellite (CLCuMuA, Accession no. MG373558) and Gossypium darwinii symptomless alphasatellite (GDarSLA, Accession no. MG373559) α-Rep. The α-Rep amino acid sequences from 19 entries in NCBI database were aligned using Maximum-Likelihood statistical method, with Jones–Taylor-Thomson (JTT) amino-acid substitution model, with 1000 bootstrap replicates. The accession number, host, geographical location, year of isolation have been mentioned for identification. The distance scale bar denotes the rate of amino acid substitution per site

Estimation of nucleotide diversity in cognate and non-cognate alphasatellite

To further understand the genetic diversity of CLCuMuA and GDarSLA alphasatellites, gene diversity (Hd) and nucleotide diversity (π) was calculated. The gene diversity (Hd) and nucleotide diversity (π) have been highlighted in bold (Table 1). CLCuMuA have higher π value (π = 0.071) as compare to GDarSLA (π = 0.053) and also have a high degree of gene diversity (Hd = 0.998) in CLCuMuA as compare to GDarSLA (Hd = 0.991). A higher level of genetic diversity in a population is generally linked with the long-term survival of the species and could potentially shape the evolutionary dynamics of a population [35,36,37].

Table 1 Genetic variability analysis of hollyhock-associated alphasatellites

Infectivity analysis of the cloned DNA of helper begomovirus and satellites

N. benthamiana plants inoculated with an agroinfectious clone of CLCuMuV-IN alone did not develop symptoms. Plants inoculated with CLCuMuV-IN and the dimeric betasatellite LuLDB developed typical symptoms of downward leaf curling, yellowing, vein thickening and stunted growth as early as 7 dpi (Fig. 4a, d, e). In contrast, co-inoculation of CLCuMuV-IN with CLCuMuA or GDarSLA delayed the appearance of the symptom by a week (Fig. 5) and the symptom produced were mild (Fig. 4b, f, g) as compared to plants inoculated with CLCuMuV-IN and LuLDB. Further, a delay of two days was observed in symptom development in plants co-inoculated with GDarSLA and the helper virus as against those co-inoculated with CLCuMuA and the helper virus (Fig. 5). The co-inoculation of betasatellite LuLDB with CLCuMuV-IN and CLCuMuA and/or GDarSLA produced severe symptoms similar to CLCuMuV-IN and LuLDB only combination however, the alphasatellites delayed the appearance of symptoms even in this case (Figs. 4, 5).

Fig. 4
figure 4

Plant infectivity assay of begomovirus and their associated satellite molecules on N. benthamiana plants. ac The phenotype of the plants inoculated with either only CLCuMuV or co-agroinoculated with satellite molecules at 7, 14 and 21 dpi, respectively. d, f, and g Close-up photograph of the plant co-agroinoculated with CLCuMuV along with GDarSLA alphasatellite and LuLDB molecules at 7, 14 and 21 dpi, respectively. Similarly, e, g and i enlarged view of the plant co-agroinoculated with CLCuMuV along with CLCuMuA alphasatellite and LuLDB molecules at 7, 14 and 21 dpi, respectively

Fig. 5
figure 5

Infectivity analysis of N. benthamiana plants inoculated with CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA in different combinations. The graph represents the symptom severity score (y-axis) vs days post-inoculation (X-axis). The table gives details of the number of symptomatic plants, dpi at which the symptoms were observed and the descriptions of the symptoms observed in each case. @ MLC; mild leaf curling, St; stunted growth, DLC; downward leaf curling, YL; yellowing of leaves, Vt; Vein thickening, SLC; severe leaf curling, ‘–’; no symptoms and $ Day of appearance of first symptoms from the date of inoculation. Symptom severity scale, 0-no symptoms, 1-very mild yellowing, 2-mild yellowing with downward leaf curling, 3-severe yellowing of leaves with downward leaf curling and vein thickening, 4-severe yellowing of leaves with downward leaf curling, vein thickening and stunted plant growth, 5-severe yellowing of leaves with downward leaf curling, vein thickening and stunted plant growth leading to plant death

Effect of alphasatellites on helper begomovirus and betasatellite replication

As discussed above, the infectivity analysis suggested that the alphasatellites suppress and delay symptom development. To further confirm these observations, we performed quantitative real-time PCR (qPCR) of plants N. benthamiana samples inoculated with CLCuMuV-IN and its associated satellite molecules in different combinations at three different time points (Fig. 6a–d). The qPCR results indicate that CLCuMuV-IN accumulates to similar levels in plants inoculated with CLCuMuV-IN alone or together with LuLDB (Fig. 6a). However, in the presence of either alphasatellites, the accumulation of CLCuMuV-IN is significantly reduced at 7 dpi (Fig. 6a, e). But no significant difference in CLCuMuV-IN levels is observed at 14 and 21 dpi (Fig. 6a, e). This observation corroborates the results of infectivity analysis wherein presence of alphasatellite the symptoms appear at 9–11 dpi as against 7 dpi observed in the case of CLCuMuV-IN and LuLDB only combination. Consistent with the infectivity analysis, no difference in CLCuMuV-IN accumulation was observed among the two alphasatellites (Fig. 6a, e). Interestingly, the presence of either alphasatellites does not affect betasatellite accumulation (Fig. 6b, f). LuLDB accumulates to similar levels in plants co-inoculated with CLCuMuV-IN + LuLDB + CLCuMuA or GDarSLA or with CLCuMuV-IN and LuLDB only (Fig. 6b, f).

Fig. 6
figure 6

Quantification (qPCR) of viral DNAs in N. benthamiana plants inoculated with CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA in different combinations at 7, 14 and 21 dpi. a Quantification of CLCuMuV-IN, b Quantification of LuLDB, c Quantification of CLCuMuA d Quantification of GDarSLA, e Comaprison of levels of CLCuMV-IN in N. benthamiana plants inoculated with CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA in different combinations at 7, 14 and 21 dpi. f Comaprison of levels of LuLDB in N. benthamiana plants inoculated with CLCuMuV-IN, LuLDB, CLCuMuA and GDarSLA in different combinations at 7, 14 and 21 dpi.The graphs represent an average of six samples for each combination. Statistical significance was calculated using ANOVA. *** indicates p value 0.0001–0.001, ** indicates p value 0.001–0.01, * indicates p value 0.01–0.05, ns ≥ 0.05 not significant


The association of CLCuMuA alphasatellite in the disease complex might give an advantage for the symptom induction in plants. Only a few geminivirus-associated satellites were reported to be associated with hollyhock plants such as Cotton leaf curl Bangalore betasatellite (CLCuBaB) [38], Cotton leaf curl Multan betasatellite (CLCuMuB), LuLDB [13], and Malvastrum yellow mosaic alphasatellite (MaYA). In recent years, the association of CLCuMuV with Malvaceae (cotton, okra, hollyhock, hibiscus species), Solanaceae (chilli, tomato), Cucurbitaceae (luffa species) family of crop plants have been extensively studied in India and Pakistan (Figs. 2, 3) [39,40,41]. However, the role of satellites in disease-complex has not been elucidated in detail.

The results of this and a previous study [18] confirm that the monopartite begomovirus CLCuMuV-IN, a betasatellite LuLDB and two alphasatellites are associated with yellow mosaic disease of hollyhock. The infectivity analysis and qPCR confirmed that CLCuMuV-IN is capable of replication and systemic infection in N. benthamiana. However, symptoms appear only when CLCuMuV-IN and its associated betasatellite, LuLDB are co-inoculated. When either CLCuMuA or GDarSLA was co-inoculated with LuLDB and/or CLCuMuV, symptoms were delayed and attenuated and the helper virus accumulated to relatively lower levels at early stages of infection (Figs. 5, 6e). The alphasatellites did not affect the accumulation of the betasatellite LuLDB (Fig. 6f).

Furthermore, variation in the begomovirus genome allows the virus population to be dynamic and might help broaden their host range [42]. A higher nucleotide diversity index (π =  > 0.07) of CLCuMuA (Table 1) further suggests that this alphasatellite contribute some advantages to the helper virus for replication. However, α-Rep of the CLCuMuA is distinctly related with the α-Rep of GDarSLA and it might modulate the helper virus replication. Previous studies have shown that several alphasatellites are capable of replicating and systemically infecting their host in the presence of a helper virus with varying effects on symptomatology and virulence. Ageratum yellow vein Singapore alphasatellite (AYVSGA) reduces symptoms severity and the relative accumulation of its associated betasatellite, Tomato leaf curl betasatellite (ToLCB), without affecting the accumulation of the helper virus [15]. In another example, Tomato yellow leaf curl China alphasatellite (TYLCCNA) reduces the accumulation of both the helper virus Tomato yellow leaf curl China virus (TYLCCNV) and its associated betasatellite Tomato yellow leaf curl China betasatellite (TYLCCNB, [16]). Delay and attenuated symptom development and reduction in titer of the helper virus DNA components were also observed when Ageratum yellow vein alphasatellite (AYVA) was co-inoculated with different cassava mosaic geminiviruses (CMGs) [43]. Interestingly, a reverse trend was observed in the case of alphasatellite associated WDIV, a mastrevirus. Here, the presence of alphasatellite increased WDIV accumulation and resulted in reduced accumulation of VsiRNA [17]. The contrasting effect of alphasatellites on helper virus and betasatellite accumulation also depends on the virus-host combination. For example, in the presence of Euphorbia yellow mosaic alphasatellite (EuYMA), the accumulation of Euphorbia yellow mosaic virus (EuYMV) DNA A increases in two plant host E. heterophylla and N. benthamiana, whereas EuYMA reduces EuYMV DNA-A levels in Arabidopsis thaliana [44]. On the contrary EuYMV DNA-B accumulation increases in the presence of EuYMA, in E. heterophylla and its levels in N. benthamiana and A. thaliana remains unchanged [44].

Suppression of silencing by α-Rep, its interaction and interference with βC1 activity, blocking the function of the helper virus Rep are some of the hypotheses put forward to explain the mechanism underlying the interaction between alphasatellites and its helper virus/betasatellite complex [13, 15, 45, 46]. Recently, potential genes regulated by TYLCCNA have been identified in a transcriptome profile [16]. Silencing of these TYLCCNA responsive genes causes severe symptoms and increased viral DNA accumulation, suggesting that these genes could contribute to host resistance against TYLCCNV/TYLCCNB infection [16].


In summary, our results demonstrate a new and differential interaction between alphasatellites CLCuMuA or GDarSLA and the associated helper virus and betasatellite complex. However, the specific molecular mechanism underlying this interaction remains to be determined. The evolutionary dynamics of α-Rep suggest further, it might have evolved from a common ancestor and then undergone selective differentiation and speciation. The higher genetic variability in CLCuMuA alphasatellite suggests that it might have acquired selective advantage during the course of evolution enabling it  to infect several families of crop plants. It has been also hypothesized that alphasatellites associated with begomoviruses might have been acquired by the helper viruses to modulate the virulence to achieved enhance virus fitness. As these interactions could be host-specific, the interaction between these viral DNAs in the natural host hollyhock remains to be determined. Based on this study, transcriptome analysis of the differentially regulated genes in plants co-inoculated with alphasatellites, the nature of the interaction of α-Rep with other viral proteins such as helper viral Rep and Transcriptional activation protein (TrAP) and cellular replisome can be examined to get molecular details of these interactions. Alpha-Rep is known to suppress TGS which could reduce viral DNA methylation allowing replication. It is tempting to speculate that α-Rep could modulate other epigenetic modifications by interacting with various cellular and viral proteins, which could, in turn, affect replication and accumulation of the helper virus. These studies could help us understand why satellite DNA molecules are acquired by the helper viruses and the biological significance of these interactions.

Availability of data and materials

All data generated or analysed during this study are included in this published article and supplementary information files. The genome sequences of the geminiviruses, betasatellite and alphasatellites are available at the GenBank (



Cotton leaf curl Multan virus-India


Cotton leaf curl Multan alphasatellite


Gossypium darwinii symptomless alphasatellite


Ludwigia leaf distortion betasatellite



MEGA-X tool:

Molecular evolutionary genetic analysis tool


Partial tandem repeat constructs


Quantitative PCR


Post-transcriptional gene silencing


Transcriptional gene silencing




Satellite conserved region




  1. 1.

    Zerbini FM, Briddon RW, Idris A, Martin DP, Moriones E, Navas-Castillo J, et al. ICTV virus taxonomy profile: geminiviridae. J Gen Virol. 2017;98:131–3.

    CAS  Article  Google Scholar 

  2. 2.

    Briddon RW, Bull SE, Amin I, Idris AM, Mansoor S, Bedford ID, et al. Diversity of DNA β, a satellite molecule associated with some monopartite begomoviruses. Virology. 2003;312:106–21.

    CAS  Article  Google Scholar 

  3. 3.

    Briddon RW, Bull SE, Amin I, Mansoor S, Bedford ID, Rishi N, et al. Diversity of DNA 1: a satellite-like molecule associated with monopartite begomovirus-DNA β complexes. Virology. 2004;324:462–74.

    CAS  Article  Google Scholar 

  4. 4.

    Lozano G, Trenado HP, Fiallo-Olivé E, Chirinos D, Geraud-Pouey F, Briddon RW, et al. Characterization of non-coding DNA satellites associated with sweepoviruses (Genus Begomovirus, Geminiviridae): definition of a distinct class of begomovirus-associated satellites. Front Microbiol. 2016;7:1–13.

    Article  Google Scholar 

  5. 5.

    Fiallo-Olivé E, Martínez-Zubiaur Y, Moriones E, Navas-Castillo J. A novel class of DNA satellites associated with New World begomoviruses. Virology. 2012;426:1–6.

    Article  Google Scholar 

  6. 6.

    Briddon RW, Mansoor S, Bedford ID, Pinner MS, Saunders K, Stanley J, et al. Identification of DNA components required for induction of cotton leaf curl disease. Virology. 2001;285:234–43.

    CAS  Article  Google Scholar 

  7. 7.

    Cui X, Li G, Wang D, Hu D, Zhou X. A begomovirus DNAβ-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. J Virol. 2005;79:10764–75.

    CAS  Article  Google Scholar 

  8. 8.

    Saunders K, Norman A, Gucciardo S, Stanley J. The DNA β satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (βC1). Virology. 2004;324:37–47.

    CAS  Article  Google Scholar 

  9. 9.

    Gopal P, Pravin Kumar P, Sinilal B, Jose J, Kasin Yadunandam A, Usha R. Differential roles of C4 and βC1 in mediating suppression of post-transcriptional gene silencing: evidence for transactivation by the C2 of Bhendi yellow vein mosaic virus, a monopartite begomovirus. Virus Res. 2007;123:9–18.

    CAS  Article  Google Scholar 

  10. 10.

    Amin I, Hussain K, Akbergenov R, Yadav JS, Qazi J, Mansoor S, et al. Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex. Mol Plant-Microbe Interact. 2011;24:973–83.

    CAS  Article  Google Scholar 

  11. 11.

    Mansoor S, Khan SH, Bashir A, Saeed M, Zafar Y, Malik KA, et al. Identification of a novel circular single-stranded DNA associated with cotton leaf curl disease in Pakistan. Virology [Internet]. 1999;259:190–9.

    CAS  Article  Google Scholar 

  12. 12.

    Saunders K, Stanley J. A nanovirus-like DNA component associated with yellow vein disease of Ageratum conyzoides: evidence for interfamilial recombination between plant DNA viruses. Virology. 1999;264:142–52.

    CAS  Article  Google Scholar 

  13. 13.

    Nawaz-ul-Rehman MS, Nahid N, Mansoor S, Briddon RW, Fauquet CM. Post-transcriptional gene silencing suppressor activity of two non-pathogenic alphasatellites associated with a begomovirus. Virology [Internet]. 2010;405:300–8.

    CAS  Article  Google Scholar 

  14. 14.

    Abbas Q, Amin I, Mansoor S, Shafiq M, Wassenegger M, Briddon RW. The Rep proteins encoded by alphasatellites restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana. Virus Dis [Internet]. 2019;30:101–5.

    Article  Google Scholar 

  15. 15.

    Idris AM, Shahid MS, Briddon RW, Khan AJ, Zhu JK, Brown JK. An unusual alphasatellite associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation. J Gen Virol. 2011;92:706–17.

    CAS  Article  Google Scholar 

  16. 16.

    Luo C, Wang ZQ, Liu X, Zhao L, Zhou X, Xie Y. Identification and analysis of potential genes regulated by an alphasatellite (TYLCCNA) that contribute to host resistance against tomato yellow leaf curl China virus and its betasatellite (TYLCCNV/TYLCCNB) infection in Nicotiana benthamiana. Viruses. 2019;11:442.

    CAS  Article  Google Scholar 

  17. 17.

    Kumar J, Kumar J, Singh SP, Tuli R. Association of satellites with a mastrevirus in natural infection: complexity of wheat Dwarf India virus disease. J Virol. 2014;88:7093–104.

    Article  Google Scholar 

  18. 18.

    Kumar M, Kumar RV, Chakraborty S. Association of a begomovirus-satellite complex with yellow vein.pdf. Arch Virol. 2020;165:2099–103.

    CAS  Article  Google Scholar 

  19. 19.

    Sangeeta, Ranjan P, Kumar RV, Bhatt BS, Chahwala FD, Yadav BK, et al. Two distinct monopartite begomovirus-betasatellite complexes in western India cause tomato leaf curl disease. Virus Res [Internet]. 2021;295:198319.

    CAS  Article  Google Scholar 

  20. 20.

    Kumari P, Singh AK, Chattopadhyay B, Chakraborty S. Molecular characterization of a new species of Begomovirus and betasatellite causing leaf curl disease of tomato in India. Virus Res [Internet]. 2010;152:19–29.

    CAS  Article  Google Scholar 

  21. 21.

    Kushwaha N, Singh AK, Basu S, Chakraborty S. Differential response of diverse solanaceous hosts to tomato leaf curl New Delhi virus infection indicates coordinated action of NBS-LRR and RNAi-mediated host defense. Arch Virol [Internet]. 2015;160:1499–509.

    CAS  Article  Google Scholar 

  22. 22.

    Chakraborty S, Vanitharani R, Chattopadhyay B, Fauquet CM. Supervirulent pseudorecombination and asymmetric synergism between genomic components of two distinct species of begomovirus associated with severe tomato leaf curl disease in India. J Gen Virol [Internet]. 2008;89:818–28.

    CAS  Article  Google Scholar 

  23. 23.

    Briddon RW, Martin DP, Roumagnac P, Navas-Castillo J, Fiallo-Olivé E, Moriones E, et al. Alphasatellitidae: a new family with two subfamilies for the classification of geminivirus- and nanovirus-associated alphasatellites. Arch Virol [Internet]. 2018;163:2587–600.

    CAS  Article  Google Scholar 

  24. 24.

    Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–9.

    CAS  Article  Google Scholar 

  25. 25.

    Vinoth Kumar R, Singh D, Singh AK, Chakraborty S. Molecular diversity, recombination and population structure of alphasatellites associated with begomovirus disease complexes. Infect Genet Evol [Internet]. 2017;49:39–47.

    CAS  Article  Google Scholar 

  26. 26.

    Rozas J, Ferrer-Mata A, Sanchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017;34:3299–302.

    CAS  Article  Google Scholar 

  27. 27.

    Shafiq M, Iqbal Z, Ali I, Abbas Q, Mansoor S, Briddon RW, et al. Real-time quantitative PCR assay for the quantification of virus and satellites causing leaf curl disease in cotton in Pakistan. J Virol Methods [Internet]. 2017;248:54–60.

    CAS  Article  Google Scholar 

  28. 28.

    Prakash V, Singh A, Singh AK, Dalmay T, Chakraborty S. Tobacco RNA-dependent RNA polymerase 1 affects the expression of defence-related genes in Nicotiana benthamiana upon Tomato leaf curl Gujarat virus infection. Planta [Internet]. 2020;252:11.

    CAS  Article  Google Scholar 

  29. 29.

    Basu S, Kumar Kushwaha N, Kumar Singh A, Pankaj Sahu P, Vinoth Kumar R, Chakraborty S. Dynamics of a geminivirus-encoded pre-coat protein and host RNA-dependent RNA polymerase 1 in regulating symptom recovery in tobacco. J Exp Bot. 2018;69:2085–102.

    CAS  Article  Google Scholar 

  30. 30.

    Malathi VG, Renukadevi P, Chakraborty S, Biswas KK, Roy A, Sivalingam PN, et al. Begomoviruses and their satellites occurring in India: distribution, diversity and pathogenesis. Singapore: Springer; 2017.

    Google Scholar 

  31. 31.

    Zubair M, Zaidi SS-A, Shakir S, Farooq M, Amin I, Scheffler JA, et al. Multiple begomoviruses found associated with cotton leaf curl disease in Pakistan in early 1990 are back in cultivated cotton. Sci Rep [Internet]. 2017;7:680.

    CAS  Article  PubMed Central  Google Scholar 

  32. 32.

    Saleem H, Nahid N, Shakir S, Ijaz S, Murtaza G, Khan AA, et al. Diversity, mutation and recombination analysis of cotton leaf curl geminiviruses. PLoS ONE [Internet]. 2016;11:1–12.

    CAS  Article  Google Scholar 

  33. 33.

    Siddiqui K, Mansoor S, Briddon RW, Amin I. Diversity of alphasatellites associated with cotton leaf curl disease in Pakistan. Virol Rep [Internet]. 2016;6:41–52.

    Article  Google Scholar 

  34. 34.

    Shakir S, Zaidi SS-A, Atiq-ur-Rehman, Farooq M, Amin I, Scheffler J, et al. Non-cultivated cotton species (Gossypium spp.) act as a reservoir for cotton leaf curl begomoviruses and associated satellites. Plants [Internet]. 2019;8:127.

    CAS  Article  Google Scholar 

  35. 35.

    Deng Y, Liu T, Xie Y, Wei Y, Xie Z, Shi Y, et al. High genetic diversity and low differentiation in Michelia shiluensis, an endangered Magnolia species in south China. Forests. 2020;11:1–15.

    Google Scholar 

  36. 36.

    Juárez M, Rabadán MP, Martínez LD, Tayahi M, Grande-Pérez A, Gómez P. Natural hosts and genetic diversity of the emerging tomato leaf curl New Delhi Virus in Spain. Front Microbiol [Internet]. 2019;10:140.

    Article  Google Scholar 

  37. 37.

    Vinoth Kumar R, Singh AK, Singh AK, Yadav T, Singh AK, Kushwaha N, et al. Complexity of begomovirus and betasatellite populations associated with chilli leaf curl disease in India. J Gen Virol. 2015;96:3143–58.

    Article  Google Scholar 

  38. 38.

    Ashwathappappa KV, Venkataravanappa V, Lakshminarayana Reddy CN, Krishna Reddy M. Molecular characterization of Tomato leaf curl virus infecting hollyhock (Alcea rosea L.) in India. Indian Phytopathol [Internet]. 2020;73:339–47.

    Article  Google Scholar 

  39. 39.

    Yogindran S, Kumar M, Sahoo L, Sanatombi K, Chakraborty S. Occurrence of Cotton leaf curl Multan virus and associated betasatellites with leaf curl disease of Bhut-Jolokia chillies (Capsicum chinense Jacq.) in India. Mol Biol Rep [Internet]. 2021;48:2143–52.

    CAS  Article  Google Scholar 

  40. 40.

    Biswas KK, Bhattacharyya UK, Palchoudhury S, Balram N, Kumar A, Arora R, et al. Dominance of recombinant cotton leaf curl Multan-Rajasthan virus associated with cotton leaf curl disease outbreak in northwest India. PLoS ONE [Internet]. 2020;15:1–19.

    CAS  Article  Google Scholar 

  41. 41.

    Mishra M, Verma RK, Marwal A, Sharma P, Gaur RK. Biology and interaction of the natural occurrence of distinct monopartite begomoviruses associated with satellites in Capsicum annum from India. Front Microbiol [Internet]. 2020;11:2366.

    Article  Google Scholar 

  42. 42.

    Xavier CAD, Godinho MT, Mar TB, Ferro CG, Sande OFL, Silva JC, et al. Evolutionary dynamics of bipartite begomoviruses revealed by complete genome analysis. Mol Ecol [Internet]. 2021;30:3747–67.

    CAS  Article  Google Scholar 

  43. 43.

    Patil BL, Fauquet CM. Differential interaction between cassava mosaic geminiviruses and geminivirus satellites. J Gen Virol. 2010;91:1871–82.

    CAS  Article  Google Scholar 

  44. 44.

    Mar TB, Mendes IR, Lau D, Fiallo-Olivé E, Navas-Castillo J, Alves MS, et al. Interaction between the New World begomovirus Euphorbia yellow mosaic virus and its associated alphasatellite: effects on infection and transmission by the whitefly Bemisia tabaci. J Gen Virol [Internet]. 2017;98:1552–62.

    CAS  Article  Google Scholar 

  45. 45.

    Briddon RW, Stanley J. Subviral agents associated with plant single-stranded DNA viruses. Virology. 2006;344:198–210.

    CAS  Article  Google Scholar 

  46. 46.

    Orozco BM, Miller AB, Settlage SB, Hanley-Bowdoin L. Functional domains of a geminivirus replication protein. J Biol Chem. 1997;272:9840–6.

    CAS  Article  Google Scholar 

Download references


The authors acknowledge Dr. R.Vinoth Kumar for his valuable suggestions.


This work was supported by a Grant from DST-FISTII (Grant Number: SLS/FISTII/SC/2017) to SC. MK is supported by the UGC NON-NET merit Scholarship provided by JNU. The funders have no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Author information




MK, FZ, SC conceived the experiments and MK and FZ performed the experiments and generated the data. MK, FZ and SC analysed the data and wrote the manuscript. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Supriya Chakraborty.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have agreed for publication in the journal.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Details of the sequences, primers and standard curves of viral DNAs used for the phylogenetic analysis and quantitative real-time PCR.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kumar, M., Zarreen, F. & Chakraborty, S. Roles of two distinct alphasatellites modulating geminivirus pathogenesis. Virol J 18, 249 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Geminivirus
  • Begomovirus
  • Alphasatellite
  • Betasatellite
  • Genetic variability
  • Disease development