MOLECULAR BASIS OF VIRUS, SATELLITE RNA AND HOST PLANT INTERACTIONS
Satellite RNAs are subviral RNAs that have little sequence homology with their helper viruses, but depend on the viral genomic RNAs for replication, encapsidation and movement. We have used a satellite RNA associated with bamboo mosaic virus (BaMV) as a model (1) to develop the satellite RNA-based plant expression vector, (2) to study the satBaMV sequence and satBaMV-encoded products involved in the satellite RNA replication and movement, and (3) to analyze the interactions between the virus, subviral RNAs and host plant that lead to virus
resistance.
GENERAL OUTLINE
BaMV, a member of the potexvirus group, consists of single-stranded and positive-sense RNA genome. Satellite RNAs of BaMV (satBaMV) is the only one found to be associated with the potexvirus group. It is a linear molecule with 836 nucleotides in length and encodes a protein of 20 kDa (P20). The open reading frame (ORF) of P20 is not essential for satBaMV replication, however, the ORF of P20 is present in all isolates of satBaMV that we have sequenced. The P20 was only transiently expressed at the early stage of infection and was mainly located in the cytoplasm and nuclei of infected cells by immunoelectron microscopy. The ORF of P20 can be replaced with the reporter gene chloramphenicol transferase. Thus, satBaMV has potentially become the satellite-based plant expression vector.
With the wide collection of satBaMV isolates domestically and abroad, we found that two isolates of satBaMV exhibit extreme differences in phenotypes. They differ greatly in their abilities to reduce the helper virus accumulation, to attenuate the virus-induced symptoms, and to move systemically in the infected host plants. Thus, these two satBaMVs provide excellent materials for studying the molecular basis of the mRNA type satellite RNA-mediated interaction with virus and host plants.
PROGRESS AND ACCOMPLISHMENTS
1. Mapping the subgenomic RNA (sgRNA) core promoter sequences by the use of satBaMV vector: Insertion of subgenomic promoter-like sequences (SGP) of BaMV capsid protein (CP) gene into the satBaMV vector gave rise to the synthesis of sgRNA of satBaMV in protoplasts of Nicotiana benthamiana and leaves of Chenopodium quinoa when co- inoculated with BaMV-V genomic RNA. By deletion analysis, we have concluded that the subgenomic promoter-like sequences of CP gene contains one core promoter sequence and two upstream and one downstream enhancers.
2. Characterization of satBaMV-encoded P20 protein: Experiments of gel retardation, UV crosslinking, and Northwestern hybridization demonstrated that the P20 is a RNA-binding protein, preferentially binding to the 5' and 3' untranslated regions (UTRs) of satBaMV. The N-terminal arginine motif of P20 is the RNA-binding domain. Furthermore, we have shown that P20 protein assists the systemic movement of satBaMV. Mutants of satBaMV which blocked the P20 gene expression did not significantly affect the accumulation level of satBaMV in the inoculated leaves, but dramatically reduced the efficiency of systemical movement in the co-inoculated Nicotiana benthamiana, indicating the important role(s) of P20 for the long-distance movement of satBaMV.
3. Identification of determinants of BSL6 satBaMV responsible for its phenotypes: By the construction of chimeric satBaMVs between BSF4 and BSL6 satBaMVs, we have mapped that the determinants are located in the 5' UTR of satBaMV. Insertion of 5' UTR of BSL6 satBaMV into the BaMV genome confers the resistance to BaMV infection in C. quinoa plants. The molecular interaction among 5' UTR of satBaMV, BaMV and host plants are under investigation.
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