Progress of research on application of VIGS vectors in fruit trees

ZHANG Xinhua; JI Nana; MIN Dedong; SHAO Shujun; LI Fujun

doi:10.13925/j.cnki.gsxb.20160167

Abstract：Virus-induced gene silencing (VIGS) is a natural defense response against the intrusion of exogenous nucleic acid in plants,which recently has been exploited as a reverse genetics tool for gene function analysis in diverse plant species.The phenomenon of RNA interference (RNAi),also known as posttranscriptional gene silencing (PTGS),underlies the method of VIGS in plants.The process of VIGS starts with plant infection with the Agrobacterium tumefaciens,which contains the recombinant viral vector carrying a fragment of the plant gene to be silenced.When the virus vectors infect the plants,the inserted gene fragment is amplified by the viral replication system,spreads systemically in the infected plants and results in the synthesis of double-stranded RNA (dsRNA).Dicer or dicer-like enzymes cleave dsRNA into small interfering RNAs (siRNAs).The siRNA single strand is then integrated into a multicomponent RNA-induced silencing complex to trigger the degradation of the recombinant RNA and the corresponding host mRNA in a homology dependent manner.Compared with the stable transformation,the VIGS approach has a number of advantages.For example,the time involved in cloning the gene of interest and analysis of the VIGS phenotype can be done within 2-3 weeks,without going through the laborious and time-consuming process of generating stable mutants.VIGS can be applied to mature plants and thus allows the ability to assess the function for genes,whose mutation (or antisensing) might be lethal in sexually propagated plants.VIGS can be use to silence members of a gene family if they have enough homology at the nucleotide level.Since VIGS does not require a full-length gene for silencing,it becomes a vigorous method to characterize the function of an expressed sequence tag.A further advantage of VIGS is its simplicity and robustness and that it is well suited to the high-throughput analysis of many genes in a target plant genome.Consequently,this facile technology is particularly important for plants with long life cycles,such as fruit trees.However,VIGS technology is limited by the host range of the virus upon which the virus vector is based.Therefore,it is the key to selecting a suitable vector for successful implementation of VIGS in a variety of fruit trees.There have been three fundamental types of viruses modified to develop VIGS vectors:RNA,DNA and satellite viruses.However,there are relatively few virus vectors for the analysis of gene functions in fruit trees due to the limitations of host range and the long growth periods of fruit trees.Currently,the VIGS vectors available for silencing studies in fruit trees mainly derived from RNA 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 above viruses,TRV is pathogenic for more than 400 plant species and able to spread more vigorously throughout the entire plant,including meristem tissue,yet the overall symptoms of infection are mild compared with other viruses.The vector based on TRV,which is widely used at present for VIGS analysis,can induce gene silencing in the leaf,flower,seedlings and fruit of several fruit trees,such as Prunus persica,Prunus avium,Malus domestica,Pyrus communis,Litchi chinensis,and Fragaria ananassa species.Therefore,the TRV-derived VIGS holds promise as a powerful tool for large-scale analysis of the functions of genes in a variety of fruit trees.ALSV,originally isolated from an apple tree,does not induce any obvious symptoms in most of host species including apple.ALSV-mediated VIGS system can effectively induce reliable silence of endogenous genes in the seedlings of apple,pear,and Japanese pear,which will be a powerful tool for functional genomics in Rosaceae fruit trees.CTV-based VIGS vector could be a useful tool for reverse genetics to study the functions of citrus genes involved in basic cellular functions,metabolic pathways,developmental biology,and plant-microbe interactions.The CLBV-based vector has also been proved useful to analyze gene function by reverse genetics in the long-lived citrus plants.Silencing of the endogenous PDS gene,which encode phytoene desaturase enzyme involved in carotenoid biosynthesis,the in grapevine plantlets has also been achieved using the GLRaV-2-derived VIGS system by inoculating seedlings,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 are limited,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 certain limitations inherent in VIGS,this technology has,to a great extent,fulfilled its promise of being a fast and efficient 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 their application 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.