A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

Abstract

Background Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. Results In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a “Y- type” asymmetric structure and contained an axillary bud that was about 1–3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. Conclusions Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

Advancing Virus-Induced Gene Silencing in Sunflower: key factors of VIGS spreading and a novel simple protocol

“…Notably, the most commonly used agroinfiltration method for VIGS is the syringe infiltration of leaves. This methods has been applied in VIGS studies on a broad range of plant species: Brassica oleracea [ 3 ], Gerbera hybrid [ 45 ], Solanum lycopersicum [ 46 ], lettuce [ 47 ], Oryza sativa [ 48 ], Triticum aestivum [ 31 ], Eschscholzia californica [ 49 ], Capsicum annuum [ 50 ], Gossypium hirsutum [ 51 ]. In sunflower [ 38 ], using syringe infiltration, the authors observed the photo-bleaching phenotype in expanded leaves and leaflets near the inflorescence.…”

Establishment of a Homologous Silencing System with Intact-Plant Infiltration and Minimized Operation for Studying Gene Function in Herbaceous Peonies

“…This reverse genetic technique adopts viral vectors carrying a target gene fragment to produce dsRNA and then triggers RNA-mediated gene silencing [16]. One of the most widely used viral delivery vectors is tobacco rattle virus (TRV) due to its simple expression in meristematic tissue [17]. VIGS has many advantages compared with other loss-of-function approaches Undoubtedly, P. lactiflora Pall.…”

“…This reverse genetic technique adopts viral vectors carrying a target gene fragment to produce dsRNA and then triggers RNA-mediated gene silencing [16]. One of the most widely used viral delivery vectors is tobacco rattle virus (TRV) due to its simple expression in meristematic tissue [17]. VIGS has many advantages compared with other loss-of-function approaches because it allows phenotypes to achieve rapid performance, provides time-saving experimental processes, does not require complex tissue culture, and has the potential to silence either individual or multiple members of a gene family [18].…”