Small interfering RNA derived from the plant virus and its application in researches on fruit tree viruses

Abstract: Fruit crops belong to perennial woody plants, which may carry viruses during lifetime once they are infected. Fruit tree-infecting viruses accumulate year by year, resulting in reduced tree vigor, fruit yield and quality, and even leading to tree death. Fruit tree virus has a wide host range and multiple transmission pathways, and its spread is accelerated with the frequent transfer of plant stocks. Moreover, fruit tree is usually simultaneously infected with multiple virus species, and the symptom is latent or not easy to be observed. Various differential hosts are usually required to distinguish fruit tree viruses that mostly lack the corresponding herbaceous host. Besides, the amounts of virus in woody plants are small, unevenly distributed, and have seasonal differences. For these reasons, the theoretical and techno- logical researches on fruit tree viruses is relatively backward. However, deep sequencing of small RNA has been successfully applied to the identification of fruit tree virus and the use of hairpin structure to express specific virus double-stranded RNA (dsRNA) for fruit trees to obtain antiviral sources has been studied. In this paper, the mechanism of RNA silencing, the origin of vsiRNA and its biosynthesis process are summarized, which emphatically elaborates the application of vsiRNA in the studies on fruit tree virus, so as to provide ideas and methods for the identification of fruit tree viruses, to acquire fruit tree antiviral resources and to cultivate virus-free stocks. RNA silencing, also known as RNA interfer- ence (RNAi), has been demonstrated as a conserved antiviral mechanism in plants. In plants, RNAi is activated by dsRNA, which is recognized by Dicer-like (DCL) proteins and cleaved into 21-24 nt small RNA. These small RNAs are loaded into RNA-induced silencing complex (RISC) containing ARGO- NAUTE (AGO) protein for post-transcriptional gene and transcriptional gene silencing. The gene silencing elements of host plants can recognize the dsRNA formed during the process of virus infection and then produce 21-24 nt small RNA, which is the virus-derived small interfering RNA (vsiRNA). Virus in- fection induces production of a great amount of vsiRNAs, which can be loaded into host AGO proteins to form RISC to target viral genome. In recent years, genetic analysis of Arabidopsis thaliana and high- throughput sequencing of vsiRNAs from various plants have revealed the origin and biosynthesis of vsiRNAs. For RNA viruses, vsiRNA is mainly derived from the dsRNA formed by the viral replication intermediate, the dsRNA region formed by the self-complementary pairing of the single stranded RNA of the viral genome, or the dsRNA produced by RNA-dependent RNA polymerase (RDR) that uses vi- ral RNA as a template to replicate. For DNA viruses, research on the source of vsiRNA is less. Until now, vsiRNA production in different plants has not been clearly illuminated, and the known vsiRNA production suggests that vsiRNA production requires the participation of multiple core components of RNA silencing, such as DCL, RDR and AGO proteins. Plant DCL proteins are critical components in the RNA silencing pathway. Different DCL proteins process dsRNA formed during viral infection into specific vsiRNA. The model plant Arabidopsis thaliana encodes four DCL proteins, DCL1, DCL2, DCL3 and DCL4, each of which has its own function. DCL1 mainly contributes to the production of miRNA and plays an indirect role in the production of different length of vsiRNA, while DCL4, DCL2 and DCL3 produce siRNA of 21-, 22- and 24-nt, respectively. Although the functions of DCL proteins in the production of vsiRNA are different, they play a complementary and self-balancing role. RDR recognizes RISC cleavage products and converts them as substrates into dsRNA, which once again enter the DCL protein containing cleavage complex to produce secondary siRNA, amplifying the silencing signal. RDR6 has been reported to be a main enzyme involved in the production of secondary small RNA in the cytoplasm. AGO protein is a key component in the RNA silencing effect complex, which binds to vsiRNA to cleave viral RNA, and the cleaved single stranded RNA is the source of secondary vsiRNA. With the development of small RNA sequencing technology, the vsiRNA expression profile of fruit tree viruses has been reported successively. Whereas, the types and functions of endogenous RDR- DCL-AGO homologues involved in vsiRNA production in fruit crops remain unclear, and the studies are rare on the small RNA-mediated antiviral gene silencing pathway in fruit trees. However, vsiRNA has been widely used in the studies on fruit tree viruses based on its origin and distribution characteris- tics. Specifically, a large amount of vsiRNA can be obtained by deep sequencing of small RNA. Since the sequences of vsiRNA are continuous and overlapped, vsiRNA can be assembled de novo into contig through bioinformatics tools for the identification of new viruses, which provides a basis for virus detec- tion. Deep sequencing of small RNA itself can also be used for molecular virus detection, which overcomes the limitations of traditional virus detection methods and can be applied to diagnose fruit tree viruses in a large scale and quickly. This technique plays an important role in virus detection and certifica- tion of virus-free plants. In addition, vsiRNA, based on deep sequencing, has also been used to study vi- rus populations in plants. Stem tip tissue culture has been widely used to obtain virus-free plants in pro- duction. Current view is that the virus-free stem tip is mainly attributed to its strong RNA silencing abil- ity, suggesting the possibility that the virus-free stem tip or the increased long virus-free stem tip of fruit trees could be obtained through expression of specific vsiRNA. Expression of vsiRNA like the hairpin RNA has been considered as a feasible technology applied to antiviral genetic engineering for fruit trees to obtain virus-resistant plants. In addition, RNAi induced by artificial miRNA in fruit tree antiviral ge- netic engineering needs to be further investigated. In conclusion, the detailed description of the applica- tion of vsiRNA in the studies on fruit tree virus will contribute to controlling viral disease of fruit trees.