Research progress in VIGS technology and its application in Cucurbitaceae crops

Abstract: Gene silencing comprises transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) . Virus-induced gene silencing (VIGS) is a natural defense reaction that exists extensively in organisms. It resists foreign nucleic acids through PTGS at RNA level. This mechanism has been explored in the development of vectors for gene verification. PTGS was first discovered in plants and described as co-suppression. Since then, it has also been found in fungi and animals, and the term was subsequently applied almost exclusively to techniques involving recombinant viruses to knock down the expression of endogenous genes. Because it can specifically silence a specific gene, leading to the loss of function of this gene, the potential of VIGS as a tool to analyze gene function is recognized quickly. Although there are many articles on the mechanism of VIGS, the specific way of action of key genes (such as the DCL gene) in the silent pathway and its effect on silencing efficiency are still not clear. Therefore, in order to better apply the VIGS technology and develop more and more optimized VIGS vectors, it is necessary to deeply study the mechanism of action of PTGS. The simplest and most effective way to determine the function of a gene or protein is to attenuate the expression of a gene or to produce a mutant that does not encode a functional protein, and the most mature technologies for studying loss of plant function are chemical mutagenesis, transposons, and innsertion of T-DNA to create disruption in the coding sequence. However, all these methods require tedious processes, which are timeconsuming and labor-intensive and have very low conversion efficiency. However, VIGS can overcome these deficiencies and be seen as an alternative approach to those traditional methods. It can identify the loss-of-function phenotype of a particular gene in a single generation. Through the use of targeting se-quences from the most highly conserved regions of the gene family, all or most of the members of a given family can be silenced to resolve complications due to the presence of gene families. It does not need to go through genetic transformation and it is a transient method. According to the latest statistics by the FAO in 2016, the output of Chinese cucurbit crops such as watermelons, melons, and cucumbers has increased yearly in the past decade and has played an important role in Chinese fruits and vegetables. China has a long history of cultivation of cucurbit crops. With the improvement of the level of agricultural production and consumers' pursuit of high-quality melon vegetables, it is urgent to utilize genes for important agronomic traits of cucurbit crops to cultivate excellent, disease-resistant and characteristic varieties. The genome sequencing of cucumber, watermelon, melon and other cucurbit crops has laid the foundation for excellent gene mining and gene function research.The genetic transformation of cucurbit crops is time-consuming and labor-intensive, and the transformation efficiency is extremely low. The establishment of the VIGS technology platform facilitates the use of VIGS technology for the study on gene function and accelerates the research of functional genomics in cucurbit crops, thus promoting the sustainable development of the cucurbit crop industry in China. Therefore, VIGS technology has a good prospect to be applied in cucurbit crops. Until now, only two VIGS vectors have been used in cucurbit crops, namely apple latent flat virus (ALSV) and tobacco ringspot virus (TRSV) based VIGS vectors.Although the VIGS system has been successfully applied in many crops as well as model plants such as Nicotiana benthamiana and Arabidopsis thaliana, some factors such as the limitation of the host range of the vector, the selection of effective target gene fragments, and the instability of the silencing efficiency are still the main concern. The construction strategy of VIGS vector and the factors affecting the efficiency of silencing are also briefly summarized. Meanwhile, the the problems that need to be solved in the future are also prospected. Developing a vector with broader host range and improving vector silencing efficiency will be a major direction for future research. Moreover, the selection of the target gene fragment for insertion into the vector also requires the exploration of more efficient fragments based on the different vectors. In addition, there are certain limitations on the location and the duration of silence, which requires further exploration. There are tremendous researches in VIGS technology. This article gives a brief overview of the development, mechanism and relative advantages of VIGS, and discusses some problems existing in the technology. The VIGS vector used in cucurbit crops is also introduced.