A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein

Abstract

Rolling-circle replication of single-stranded genomes of plant geminiviruses is initiated by sequence-specific DNA binding of the viral replication-related protein (Rep) to its cognate genome at the replication origin. Monopartite begomovirus-associated betasatellites can be trans replicated by both cognate and some noncognate helper viruses, but the molecular basis of replication promiscuity of betasatellites remains uncharacterized. Earlier studies showed that when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) is coinoculated with both cognate and noncognate betasatellites, the cognate betasatellite dominates over the noncognate one at the late stages of infection. In this study, we constructed reciprocal chimeric betasatellites between tomato yellow leaf curl China betasatellite and tobacco curly shoot betasatellite and assayed their competitiveness against wild-type betasatellite when coinoculated with TYLCCNV or TbCSV onto plants. We mapped a region immediately upstream of the conserved rolling-circle cruciform structure of betasatellite origin that confers the cognate Rep-mediated replication advantage over the noncognate satellite. DNase I protection and in vitro binding assays further identified a novel sequence element termed Rep-binding motif (RBM), which specifically binds to the cognate Rep protein and to the noncognate Rep, albeit at lower affinity. Furthermore, we showed that RBM-Rep binding affinity is correlated with betasatellite replication efficiency in protoplasts. Our data suggest that although strict specificity of Rep-mediated replication does not exist, betasatellites have adapted to their cognate Reps for efficient replication during coevolution. IMPORTANCEBegomoviruses are numerous circular DNA viruses that cause devastating diseases of crops worldwide. Monopartite begomoviruses are frequently associated with betasatellites which are essential for induction of typical disease symptoms. Coexistence of two distinct betasatellites with one helper virus is rare in nature. Our previous research showed that begomoviruses can trans replicate cognate betasatellites to higher levels than noncognate ones. However, the molecular mechanisms of betasatellites selective replication remain largely unknown. We investigated the interaction between the begomovirus replication-associated protein and betasatellite DNA. We found that the replication-associated protein specifically binds to a motif in betasatellites, with higher affinity for the cognate motif than the noncognate motif. This preference for cognate motif binding determines the selective replication of betasatellites. We also demonstrated that this motif is essential for betasatellite replication. These findings shed new light on the promiscuous yet selective replication of betasatellites by helper geminiviruses. Viruses of the family Geminiviridae have small circular singlestranded DNA (ssDNA) genomes encapsidated within geminate particles. They are divided into seven genera based on their genome or...

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

“…Arabidopsis thaliana ecotype Col‐0 and Nicotiana benthamiana plants used in this study were grown as previously described (Zhang et al ., , ). Agro‐infectious clones of TMV‐GFP (Shivprasad et al ., ) and CMV (Fny strain) (Liao et al ., ) and were kindly provided by Prof. Xueping Zhou (Zhejiang University) and Dr. Zhouhang Gu (Zhejiang Sci‐Tech University), respectively.…”

Iterons Homologous to Helper Geminiviruses Are Essential for Efficient Replication of Betasatellites

“…Analysis of the satellite DNAs revealed that the dRBM mutant was unable to accumulate to a detectable level (Fig. 1B, lanes 15 and 16), suggesting an important role for the RBM sequence in betasatellite replication (40). The dIte I mutant accumulated to a considerably lower level than WT Y35␤ (Fig.…”

“…Specific Rep-iteron binding confers a cognate helper virus-mediated replication preference. The TYLCCNB-[Y10] betasatellite (abbreviated as Y10␤) associated with TYLCCNV-[Y10] (abbreviated as Y10) contains an RBM sequence in a position analogous to that in Y35␤ (40), and within the RBM, two iteron-like repeated sequence elements (5=-GGGACACC-3=) were identified (Fig. 3A).…”

“…In addition, phylogenetic analysis has revealed a linear correlation between pairwise nucleotide sequence identities of betasatellites and helper begomoviruses, suggesting that these satellite molecules have coevolved and adapted to their cognate helper viruses and that reassortment events may not be prevalent (6). In laboratory experiments, when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) were coinfected with both of their associated betasatellites, i.e., tomato yellow leaf curl China betasatellite (TYLCCNB) and tobacco curly shoot betasatellite (TbCSB), only the cognate satellite relative to the supporting helper virus was stably maintained at late stages of infection (38,40). Accordingly, despite the apparent replication promiscuity, simultaneous association of two distinct betasatellites with a single helper begomovirus isolate has rarely been reported in the field.…”

“…We previously mapped a short region upstream of the SCR, called the Rep-binding motif (RBM), in TYLCCNB and TbCSB that is critical for specific Rep binding and in vivo replication, as well as for cognate virus-mediated preferential replication (40). In this study, using TbCSB as a model, we identify two tandem repeats (iterons) in the RBM that are required for specific Rep binding and efficient replication, and we show that identical iteron sequences present in the TbCSV are also necessary for Rep binding and helper virus replication.…”