Progress of research on application of VIGS vectors in fruit trees

Abstract: Virus-induced gene silencing (VIGS) is a natural defense response against the intrusion of ex⁃ogenous nucleic acid in plants, which recently has been exploited as a reverse genetics tool for gene func⁃tion analysis in diverse plant species. The phenomenon of RNA interference (RNAi), also known as posttranscriptionalgene silencing (PTGS), underlies the method of VIGS in plants. The process of VIGS startswith plant infection with the Agrobacterium tumefaciens, which contains the recombinant viral vector carry⁃ing a fragment of the plant gene to be silenced. When the virus vectors infect the plants, the inserted genefragment is amplified by the viral replication system, spreads systemically in the infected plants and re⁃sults in the synthesis of double-stranded RNA (dsRNA). Dicer or dicer-like enzymes cleave dsRNA intosmall interfering RNAs (siRNAs). The siRNA single strand is then integrated into a multicomponentRNA-induced silencing complex to trigger the degradation of the recombinant RNA and the correspond⁃ing host mRNA in a homology dependent manner. Compared with the stable transformation, the VIGS ap⁃proach has a number of advantages. For example, the time involved in cloning the gene of interest andanalysis of the VIGS phenotype can be done within 2-3 weeks, without going through the laborious andtime-consuming process of generating stable mutants. VIGS can be applied to mature plants and thus al⁃lows the ability to assess the function for genes, whose mutation (or antisensing) might be lethal in sexual⁃ly propagated plants. VIGS can be use to silence members of a gene family if they have enough homologyat the nucleotide level. Since VIGS does not require a full-length gene for silencing, it becomes a vigorousmethod to characterize the function of an expressed sequence tag. A further advantage of VIGS is its sim⁃plicity and robustness and that it is well suited to the high-throughput analysis of many genes in a targetplant genome. Consequently, this facile technology is particularly important for plants with long life cy⁃cles, such as fruit trees. However, VIGS technology is limited by the host range of the virus upon whichthe virus vector is based. Therefore, it is the key to selecting a suitable vector for successful implementa⁃tion of VIGS in a variety of fruit trees. There have been three fundamental types of viruses modified to de⁃velop VIGS vectors: RNA, DNA and satellite viruses. However, there are relatively few virus vectors forthe analysis of gene functions in fruit trees due to the limitations of host range and the long growth periodsof fruit trees. Currently, the VIGS vectors available for silencing studies in fruit trees mainly derived fromRNA viruses such as tobacco rattle virus (TRV), apple latent spherical virus (ALSV), citrus tristeza virus(CTV), citrus leaf blotch virus (CLBV) and grapevine leafroll-associated virus (GLRaV). Among the aboveviruses, TRV is pathogenic for more than 400 plant species and able to spread more vigorously throughoutthe entire plant, including meristem tissue, yet the overall symptoms of infection are mild compared withother viruses. The vector based on TRV, which is widely used at present for VIGS analysis, can inducegene silencing in the leaf, flower, seedlings and fruit of several fruit trees, such as Prunus persica, Prunusavium, Malus domestica, Pyrus communis, Litchi chinensis, and Fragaria ananassa species. Therefore, theTRV-derived VIGS holds promise as a powerful tool for large-scale analysis of the functions of genes in avariety of fruit trees. ALSV, originally isolated from an apple tree, does not induce any obvious symptomsin most of host species including apple. ALSV-mediated VIGS system can effectively induce reliable si⁃lence of endogenous genes in the seedlings of apple, pear, and Japanese pear, which will be a powerfultool for functional genomics in Rosaceae fruit trees. CTV-based VIGS vector could be a useful tool for re⁃verse genetics to study the functions of citrus genes involved in basic cellular functions, metabolic path⁃ways, developmental biology, and plant-microbe interactions. The CLBV-based vector has also beenproved useful to analyze gene function by reverse genetics in the long-lived citrus plants. Silencing of theendogenous PDS gene, which encode phytoene desaturase enzyme involved in carotenoid biosynthesis, thein grapevine plantlets has also been achieved using the GLRaV-2-derived VIGS system by inoculatingseedlings, thereby providing a method and an important tool for reverse genetic studies of a variety of processes occurring in Vitis vinifera. Although the current virus vectors for VIGS analysis in fruit trees arelimited, the number of fruit trees species amenable to VIGS experiment will be increasing with the development of newly designed virus vectors and the improvement of plant infection methods. Despite certainlimitations inherent in VIGS, this technology has, to a great extent, fulfilled its promise of being a fast andefficient functional genomics tool and will undoubtedly play a significant role in the study of the gene functions of fruit trees. This review provided an update on the most recent advances of VIGS vectors and theirapplication in the gene function analysis in fruit trees. Meanwhile, the problems and its solutions of the application of VIGS in fruit trees were also analyzed.