Characterization of alphasatellites associated with monopartite begomovirus/betasatellite complexes in Yunnan, China

Geminiviruses are a group of plant viruses characterized by their geminate shape and the size of their particles, which encapsidate a circular single-stranded DNA genome. Due to their wide host range and high frequency of genome variation, geminiviruses cause substantial yield losses in many crops, including tomato, cassava, and cotton, throughout tropical and sub-tropical regions worldwide [1, 2]. The majority of geminiviruses described belong to the genus Begomovirus in the family Geminiviridae, they are transmitted by the whitefly, Bemisia tabaci[3]. Most begomoviruses have 2 components, which are referred to as DNA-A and DNA-B, both are essential for virus proliferation. Many species only have a single genomic component that resembles DNA-A [1, 3]. Some monopartite begomoviruses are associated with betasatellites (formerly DNAβ), which affect the replication of their respective helper begomoviruses and alter the symptoms induced in some host plants [4‐9]. Analysis of betasatellites reveals that they are approximately half the size of the genomic DNA, and except for a conserved hairpin structure and a TAATATTAC loop sequence, they have little sequence similarity to either the DNA-A or DNA-B molecules of begomoviruses. Betasatellites require begomoviruses for replication, encapsidation, insect transmission, and movement in plants [10].

Alphasatellites (formerly DNA1) are circular, single-stranded DNA molecules associated with begomovirus/betasatellite complexes [11‐15]. Alphasatellites are approximately half the size of begomovirus DNA and encode a rolling-circle replication initiator protein similar to nanoviruses. Consequently, alphasatellites are capable of self-replication in host plants, but require helper begomoviruses for movement in plants as well as insect transmission.

In China, several begomoviruses are reported to infect squash, tobacco, ageratum, tomato, and malvastrum; many begomovirus isolates are associated with betasatellites, and co-evolution of betasatellites with their helper viruses has been documented [9, 16‐21]. In this report, we identify 33 alphasatellites from Yunnan Province, China, and demonstrate that they can be classified into 3 types--each associated with a specific begomovirus/betasatellite complex.

Two single-stranded DNA components, alpha- and betasatellites, have been found to be associated with monopartite begomoviruses such as AYVV, CLCuMV, and TbCSV [4, 8, 11, 13, 23]. Betasatellites are symptom-modulating satellite molecules that depend on a helper virus for proliferation and movement. On the other hand, alphasatellites are apparently dispensable for symptomatic induction and are capable of autonomous replication [12‐14]. Our results show that the 33 alphasatellites investigated are all associated with begomovirus/betasatellite complexes, which is a similar result to a report by Briddon [11]. However, only some begomovirus/betasatellite complexes were associated with alphasatellites. A better understanding of the relationship between alphasatellites and begomovirus/betasatellite complexes is achievable if future studies concentrate on the identification of alphasatellites from more symptomatic and asymptomatic crop species as well as diverse, agriculturally unimportant plant species from broader areas.

With the exception of Y89A, comparison of alphasatellites shows that they can be divided into 3 types. Type 1 alphasatellites were identified in samples infected by TYLCCNV/TYLCCNB and TYLCCNV/TYLCTHB + TYLCTHV. Since no alphasatellites were found in samples infected by TYLCTHV/TYLCTHB, it is evident that type 1 alphasatellites are associated with TYLCCNV/TYLCCNB. All type 2 alphasatellites were identified in samples infected by TbCSV/TbCSB, TbCSV/TbCSB + TbLCYNV, TbCSV/TbCSB + TYLCCNV, and TbCSV + TYLCCNV/TYLCCNB. Because no alphasatellites were found in samples infected by TbLCYNV alone, this suggests that type 2 alphasatellites are associated with TbCSV/TbCSB complexes. It is interesting that alphasatellites in sample Y146, which was mix-infected by TbCSV and TYLCCNV/TYLCCNB, were clustered in type 2 but not type 1. Sample Y146 might have been mix-infected by TYLCCNV/TYLCCNB and TbCSV/TbCSB in addition to alphasatellites associated with TbCSV/TbCSB, TbCSB then disappeared due to competition between TYLCCNB and TbCSB [24]. Most type 3 alphasatellites were found in samples mix-infected by a combination of TbCSV/AYVB and TbLCYNV or MYVYNV/MYVYNB, while 2 type 3 alphasatellites were mix-infected by TbCSV and MYVV/MYVB. Because no alphasatellites were found in samples infected by TbLCYNV, MYVYNV/MYVYNB, or MYVV/MYVB, it is apparent that type 3 alphasatellites are associated with TbCSV/AYVB. Although AYVCNV/AYVB is responsible for ageratum yellow vein disease in Hainan, China [21], AYVCNV was not found in any ageratum yellow vein disease samples in Yunnan. Instead of AYVCNV/AYVB, TbCSV/AYVB is the causal agents of ageratum yellow vein disease (Zhou et al., unpublished). It is probable that TbCSV acquires the heterogenous betasatellite, AYVB, during mixed infections, but we were unable to determine the origin of type 3 alphasatellites in this study. Sample Y89 was infected by TYLCCNV/TYLCCNB, therefore, its alphasatellite should belong to type 1. However, sequence comparison shows that Y89A shares only 69.3-79.5% nucleotide sequence identity with other alphasatellites of the 3 types. We speculate that Y89A originated from an unidentified begomovirus/betasatellite complex.

Mix-infections of begomoviruses are common; 16 of 31 isolates in this study were co-infected by 2 begomoviruses (Table 2). For most isolates, each begomovirus is associated with an alpha- and betasatellite. Two type 1 alphasatellites (Y8A-5 and Y8A-6) were identified in sample Y8, while 2 types of alphasatellites (Y87A-7 and Y87A-2) were identified in sample Y87. Our results indicate that mix-infections of alphasatellite molecules may not be unusual.

The origin of alphasatellites is undoubtedly related to nanoviruses. Presently, the function of alphasatellites is not clear, but it is evident that alphasatellites functionally interact with geminivirus/betasatellite complexes resulting in symptom alteration and a reduction in the level of viral DNA and betasatellites [12‐14, 25, 26] Available evidence suggests that the ubiquitous association of alphasatellites with begomovirus/betasatellite complexes indicates that alphasatellites may play an important role in the occurrence, diffusion, and epidemiology of begomovirus/betasatellite complexes. More studies are required to elucidate the specific role that alphasatellites play in disease development, virus life cycle, and the evolution of begomoviruses/betasatellite complexes.

Seven viruses, including TbCSV, TbLCYNV, TYLCCNV, TYLCTHV, MYVV, MYVYNV, and SLCYNV, were characterized in Yunnan Province--some of them are associated with betasatellites. Our results show that all samples from Yunnan that contained alphasatellites also had betasatellites. However, only some samples that contained betasatellites had alphasatellites. Thirty-three sequenced alphasatellites were divided into 3 types--each associated with a specific begomovirus/betasatellite complex. Type 1 was associated with TYLCCNV/TYLCCNB; type 2 was associated with TbCSV/TbCSB; and type 3 was associated with TbCSV/AYVB. Alphasatellites have 3 highly conserved structure features: a conserved hairpin structure, a single open reading frame, and an A-rich region. The alignment of the sequences of the conserved hairpin structure and the A-rich region shows that the alphasatellites can be further divided into 3 types in accordance with the phylogenetic trees of their complete nucleotide sequences. Reps encoded by the 33 alphasatellites are highly conserved and share more than 86.3% amino acid sequence identity. Alphasatellites may play an important role in the epidemiology of begomovirus/betasatellite complexes.