Analysis and application of cis-acting elements and defective RNA of Pitaya virus X
Date Issued
2014
Date
2014
Author(s)
Lee, Chih-Ching
Abstract
Pitaya, a climbing succulent plant in the family Cactaceae, has gradually become an important economic fruit in Taiwan. However, its cultivation is restricted by viral diseases. Cactus virus X (CVX), Zygocactus virus X (ZyVX) and Pitaya virus X (PiVX) have been reported to infect pitaya in Taiwan. These viruses are commonly found in the field. PiVX is a new member of the Potexvirus genus identified in our lab. We extracted total RNA from infected pitaya and discovered some small RNAs reacting to PiVX probe by Northern blot. After cloning and sequence analysis, we confirmed that these small RNAs are defective RNAs (dRNAs) of PiVX. dRNAs are subviral RNAs produced from RNA virus via deletion and recombination. dRNAs depend on their parental virus (helper virus) to replicate, encapsidate and move in host plant. To select the PiVX dRNAs with best infectivity, transcripts of different dRNA clones from pitaya were analyzed with the help of PiVX transcripts in Nicotiana benthamiana protoplasts by Northern blot. After several independent inoculation assays, we found P1 dRNA, which contains 5’ 645 nt region and 3’ 196 nt region with its total length of 841 nucleotides had the best replication ability. In order to develop PiVX dRNA as a virus expression vector, we used P1-based vector to express enhanced green fluorescent protein (EGFP) gene by two different ways and thus constructed P1-EF and P1-E. P1-EF combined the open reading frame of RNA-dependent RNA Polymerase (RdRP) with foreign gene to form a fusion protein. P1-E recruited the subgenomic promoter (SGP) of PiVX into P1 dRNA to express foreign gene through sgRNA expression。According to experimental results, P1-E could successfully express EGFP in N. benthamiana protoplasts and Chenopodium quinoa plants, but the expression efficiency was low. To improve the expression efficacy of P1-E, we tried to express p19, a gene silencing suppressor, in N. benthamiana and C. quinoa plants. The results indicated p19 had no significant effect on the expression of EGFP in C. quinoa plants. However, when P1-E was inoculated to N. benthamiana plant after transient expression of p19, PiVX CP accumulation increased but EGFP expression could not be detected by GFP antibody. In addition, in order to study the cis-acting elements that can be recognized and replicated by PiVX, we constructed and analyzed some deletion mutants of P1 dRNA in N. benthamiana protoplasts. The current result indicated that the dRNA containing only 5’ 435 nt region and 3’196 nt region could still be replicated by helper virus. Furthermore, we also cloned CVX dRNAs from pitaya plant, which contain CVX 5’ 677 nt region and 3’ 591 nt region with its total length of 1268 nucleotides. For testing the biological activity, transcripts of CVX dRNAs together with PiVX were inoculated to N. benthamiana protoplasts, and Northern blot demonstrated CVX dRNAs could be replicated by PiVX. Accordingly, the result suggested that PiVX RdRP can recognize the cis-acting elements of CVX dRNAs.
Subjects
紅龍果X病毒
仙人掌X病毒
cis-acting elements
缺失性RNA
病毒載體
次基因啟動子
Type
thesis
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