A recombinant DNA‐satellite associated with Pepper yellow leaf curl Indonesia virus in highland area

Yellow curl disease caused by Begomovirus is a major threat for horticulture in Indonesia. Control mea‐ sures for the disease face several constraints, one of which is the association between begomovirus and DNA satellites which can affect the severity of symptoms. In this study, we detected the presence of a DNA satellite associated with begomovirus in a highland area. The sample was obtained from Ketep, Magelang, located approximately 1400 meters above sea level. Begomovirus was detected using primers PAL1V1978/PAR1C715 that resulted in an amplicon of ap‐ proximately 1600bp. The presence of this satellite was detected using primers CLB36F/CLB37R, resulting in full‐length satellite genome of approximately 1300bp. Sequence analysis showed the sample was infected by Pepper yellow leaf curl Indonesia virus (PepYLCIV) and a non‐coding satellite which resembled some characteristics of common betasatellites with imperfect putative ORF βC1. SimPlot analysis revealed the recombination event between betasatellites and DNA‐B of PepYLCIV. The satellite found in this study is thought to be the result of recombination due to multiple infections in plants.


Introduction
Since 1999, the incidence of yellow curl disease has been a major threat for the chili cultivation and other Solanaceae in Indonesia (Hidayat et al. 1999; Sulandari et al. 2001. Yellow curly disease on chili in Indonesia is mainly caused by Begomovirus infection. There are several species of Begomovirus that cause chili yellow curl diseases i.e., Pepper yellow leaf curl Indonesia virus (PepYLCIV), Tomato leaf curl Kanchanaburi virus (TYL CKaV), Tomato leaf curl Java virus (ToLCJaV), Agera tum yellow vein virus (AYVV) (Kenyon et al. 2014; Wil isiani et al. 2014. Diseases caused by begomovirus in fection initially only occurred in the lowlands. But in re cent years, the disease is also found in the highlands area (Kusumaningrum et al. 2017). Besides, diseases caused by begomovirus have also been reported affecting new hosts, including eggplant and luffa (Kintasari et al.; Giovanni et al. 2020). Until now, controlling measures for yellow curl diseases are still difficult because of several factors, including a broad range of hosts, high genetic diversity, the polyphagous insect vector Bemisia tabaci, multiple in fections by several species, recombination among species, and the presence of satellite DNA which potentially affect the symptoms.
Begomovirus is a circular singlestranded DNA virus that belongs to the family Geminiviridae. It may pos sess single genome (monopartite) which only DNAA present in a single virion, but also two genomes (bipar tite) which consist of DNAA and DNAB in a single virion. The Begomovirus genome sized approximately 2.72.8kbp. The monopartite begomoviruses found in In donesia are AYVV, ToLCV, and ToLCJaV. The bipartite begomoviruses are TYLCKaV, Tomato yellow leaf curl New Delhi virus (ToLCNDV), and PepYLCIV (Kenyon et al. 2014; Tsai et al. 2006). In addition to the two genomes, begomovirus could also be associated with DNA satellites. Several monopartite Begomovirus i.e., AYVV, ToLCJaV, and Cotton leaf curl virus (CuLCV), and the bipartite ToLCNDV and TYLCKaV were associated with DNA satellites (Kon et al. 2006; Saunders et al. 2004; Nawazul Rehman et al. 2009; Agnihotri et al. 2018; Kan dito et al. 2020).
There are three types of satellites being reported to be associated with Begomovirus, alphasatellite, betasatellite, and deltasatellite. Recently, these satellites are grouped into two families Tolecusatellitidae and Deltasatellitidae (Zhou 2013; Lozano et al. 2016; FialloOlivé and Navas Castillo 2020. The presence of satellites could affect symptom sever ity. In example, betasatellites were reported to exacerbate the symptom, while alphasatellites were vice versa (Dry et al. 1997; Briddon et al. 2001; Zhang et al. 2012; Zhou 2013; Idris et al. 2011. Meanwhile, deltasatellites which sized approximately only half of their counterparts, con sisted of noncoding region and were also reported to re duce symptom severity (Zhou 2013). In this study, we aimed to detect the presence of satellite associated with se vere symptoms of the yellow disease on chili in the high land area and analyze the molecular characteristic of the satellite.

Sampling location
Leaf sample was collected from chili plantation in Ketep, Magelang, Central Java, located approximately 1400 masl. Sampling was conducted in 2018. Ketep was chosen as a sampling site because it is one of the horticulture produc tion areas in Central Java and reported as endemic area of begomovirus infections, as reported by Kusumaningrum et al. (2017). Symptom appearance of the sample was mo saic, leaf yellowing, stunting, which are common symp toms of begomovirus infection ( Figure 1).

DNA Extraction
A hundred milligrams of fresh sample was used for total DNA extraction. The sample was ground using porcelain mortar and pestle without liquid nitrogen. The DNA ex traction protocol was carried out using a total DNA extrac tion kit for plants (Geneaid, Germany). Extraction steps were done following manufacturer instructions. Total ge nomic DNA was subsequently used as a template for am plification.

Detection and amplification using PCR
Polymerase Chain Reaction (PCR) was carried out using MyTaq Redmix Polymerase (Bioline), ddH 2 O, forward primer, reverse primer, and DNA templates. The reac tion was carried out in a volume of 50 µL. The primer sequences used in this study were in Table I. The PCR pro gram for the primary pair PALIV1978/ PAR1C715 which amplified the Begomovirus partial DNAA region, i.e., predenaturation at 95°C for 3 min, followed 40 cycles of denaturation at 95°C for 1 min, attaching (annealing) at 55°C for 30 s, extension for 1 min 30 s. The final ex tension was at 72°C for 10 min. The PCR program for the CLB36F/CLB36R primer amplified the full length of be tasatellite consists of predenaturation at 95°C for 3 min, followed 40 cycles of denaturation at 95°C for 1 min, an nealing at 58°C for 30 s, extension for 1.5 min. The final extension was at 72°C for 10 min. Visualization of PCR products was carried out by agarose gel electrophoresis 1% (w / v) using 50 V power for 50 min.

Sequencing and sequence analysis
Sequence analysis of begomovirus was conducted using the direct Sanger sequencing method of PCR products. The sequence of the betasatellite was performed by the cloning method using the T7 plasmid. Analysis of se quence results was carried out with the MEGA v.7 pro gram (Kumar et al. 2016), BLAST (http://blast.ncbi.nlm .nih.gov/Blast_cgi), Clustal Omega (http://ebi.ac.uk/Tool s/msa/clustalo/), and ORFFinder (https://www.ncbi.nlm .nih.gov/orffinder/). The phylogenetic analysis uses the NeighborJoining method with 1000 bootstraps (Efron et al. 1996). The recombinant analysis was performed using Simplot v3.5.1. (Lole et al. 1999). Analysis of unique structures was performed using alignment analy sis. Subsequently, the satellite sequence was submitted to Genbank with accession number MN905560. Satellites genome diagrams were created using BioRender (http: //biorender.com).

Result
Chili sample found in the Ketep area, Magelang show ing several symptoms including mosaic, dwarfism, vein enation, and curling which are typical symptoms of Bego movirus infection in chilies. Begomovirus detection was carried out by PCR method using primer pair PALIV1978 / PARIC715 which amplifies partial DNAA. PCR re sults showed positive results for DNA bands measuring 1600bp (Figure 2a,). The detection of betasatellites was The results of the begomovirus PCR sequence showed that the sample was infected with PepYLCIV with a ho mology percentage up to 100% compared to the PepYL CIV sequences obtained from Genbank. Phylogenetic analysis using NeighborJoining with 1000 boorstraps shows the sample is in the Indonesian PepYLCIV clus ter (Figure 3). PepYLCIV is a species of begomovirus commonly found in Indonesia and has been reported to be a major problem in Solanaceae cropping since 1999. It was also reported that PepYLCIV is a begomovirus which is endemic in Indonesia. The incidence of yellow leaf curl caused by PepYLCIV in various Solanaceae can reach 100%, with the value of losses varying depending on the time of infection.
The nucleotide sequence of betasatellite shows that the satellite accompanies the infection resembling betasatel lites which associated with ToLCV and AYVV, and has similarities to the noncoding region part of DNAB on PepYLCIV. Phylogenetic analysis using the Neighbor joining method shows that this satellite is located in a clus ter with satellites associated with ToLCV and AYVV, and has proximity to the noncoding satellite previously re ported to be associated with TYLCKaV (Kandito et al. 2020) (Figure 4). The sequencing and characterization results of the satellite showed a noncoding satellite that was thought to be a recombination of betasatellite with PepYLCIV DNAB. This satellite has an SCR resem bling ToLCB, an imperfect ORF βC1, and a similarity to PepYLCIV DNAB intergenic region spanning between ORF BV1 and BC1 .
Further analysis of satellite sequences was carried out by looking at the sequence profile on 3 main parts of the satellite, namely the satellite conserved region (SCR) which contains the stemloop structure TAATAT  TAC, ORF βC1, and adenine rich region. The alignment in the SCR and stemloop regions has significant similar ities to the sequences obtained from Genbank ( Figure 5). The similarities in the SCR area indicated that the satel  Figure 5) and the presence of several stop codons located in that region. This resulted in an imperfect ORF of βC1 in the sample and it was a noncoding region ( Figure 6). Schematic diagram of the noncoding satellite compared with common betasatellite shown in Figure 7.
The percentage of adenine in the Adeninrich region reaches 66.67%. This section presents a secondary stem loop structure along 24 nucleotides TCGATTAACATA CACGTTAATCGA which can also be found in the non DNAB coding PepYLCIV in Java (AB213599). Similar parts between the satellite and its helper virus indicate the Recombination event was determined using SimPlot program v3.5.1 with a comparison between betasatellites, PepYLCIVnon coding satellite, and PepYLCIV DNAB (Figure 8). The result showed that the PepYLCIVnon coding satellite was a possible recombinant between be tasatellites and PepYLCIV DNAB. This leads to a presup position that mixed infection between begomoviruses and satellites may occur in the field, as the whitefly, the spe cific vector of begomovirus could contain more than one species of begomovirus (Sidik et al.; Purwoko et al. 2015). The recombination phenomenon between begomoviruses and their satellites was reported by Huang et al. (2013).

Discussion
The virussatellite complex that infects a plant can result in recombination between viruses and satellites, satellites, and other satellites, giving rise to defective DNA (dfDNA / diDNA), or transreplication. Transreplication is a virus that can acquire and reproduce satellites from other viruses (Zhou 2013; Nawazul Rehman et al. 2009). In this study, the secondary stemloop structure that was found in the noncoding satellite was also found on satellites associ ated with the group Sweepovirus and recombinant satel lites (Huang et al. 2013; Lozano et al. 2016). The inci dent so far could have several consequences namely ex acerbating symptoms, relieving symptoms, not affecting symptoms at al. or generating new satellites. (Huang FIGURE 7 Schematic diagram of the non-coding satellite genome associated with PepYLCIV compared with common betasatellite genome. The figure was created with BioRender (http://biorender.com). Cabbage leaf curl virus (CbLCuV), resulting in a betasatel lite progeny that could be transreplicated with other bego moviruses from the New World group. Incidence of natu ral recombination between ToLJaV, AYVV, and betasatel lites into new satellites suspected to be due to mixed infec tion of several viruses in a plant (Kon et al. 2006; Nawazul Rehman et al. 2009; Huang et al. 2013. These phenomena were apparently the result of complex, multiple infections of viruses on a single host. Several reports indicated that begomovirus, betasatellite, and alphasatellite could have coexistence due to shared genome similarity (Huang et al. 2013; Emmanuel et al. 2020. Thus, each component has a similar probability to resulted in recombination or a de fective genome.
Meanwhile, the conserved region of the bego moviruses and their satellites, which contain TAATAT TAC motifs, indicated similarities between the virus and the satellite. The TAATATTAC also served as the ori gin of replication for both virus and satellites (Yang and Zhou 2017). On the virus side, the origin of replication belongs to both DNAA and DNAB, especially served for maintaining DNAB, since DNAB has no Rep gene and it could not replicate autonomously. The DNAB replica tion depending on the Rep gene activity on the DNAA. This has also happened with the replication of betasatel lites (Yang and Zhou 2017). Briddon et al. (2010) hypoth esized that the DNAB might result from wild satellites, which apparently integrated as the second genome of be gomovirus. On the other hand, betasatellite could replace the DNAB component by facilitating systemic infection and cellular movement through the βC1 gene expression. Thus it can be one of the pathogenicity determinants for the virus infection (Saeed et al. 2007; Saunders et al. 2004; Briddon and Stanley 2006. In this study, the noncoding satellite is presumed to be the result of coinfection between PepYLCIV, a bipar tite begomovirus, with another begomovirus with satellite. The BLASTn result indicated that the satellite is appar ently a recombinant satellite. Phylogenetic results of be tasatellites showed the satellite in this study grouped with ToLCVassociated betasatellites, including noncoding satellite associated with TYLCKaV (MN510674), and ToLCPhVassociated betasatellites.
Further analysis using SimPlot v3.5.1. based on nucleotide alignment indicated the recombination oc curred between a betasatellite with the DNAB compo nent of PepYLCIV. The recombination itself is common among begomoviruses or begomovirussatellites, since each genome shared a similar origin of replication, and both DNAB and betasatellites are dependent on DNAA for their maintenance (Huang et al. 2013). However, the occurrence of begomovirusbetasatellite recombinations is different from the occurrence of defective DNA (dfDNA) of begomovirus (Tahir et al. 2017). The dfDNA is the result of incomplete replication, thus creating significant deletions of the genome, while recombination is likely to be a result of transreplication (Nawazul Rehman et al. 2009).

Conclusions
The chili sample we collected from the highland area was infected by PepYLCIV with a DNA satellite. The satellite found in this study have similar characteristic with other common betasatellites including the common stemloop region, adenine rich region, sized around 1.3 kbp, except imperfect predictive ORF which considering the satellite is a noncoding. The noncoding satellite DNA found to be associated with PepYLCIV in this study is thought to be the result of recombination due to multiple infections in plants. Further, it is necessary to examine the role of this noncoding satellite in understanding the evolution of begomovirus and its satellites.