A tobacco ringspot virus-based vector system for gene and microRNA function studies in cucurbits

Abstract

Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and non-coding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.

A novel strategy for improving watermelon resistance to cucumber green mottle mosaic virus by exogenous boron application

“…To circumvent this obstacle, we decided to investigate the molecular mechanisms controlling CGMMV infection in watermelon fruits through reverse genetics using a CGMMV‐based virus‐induced gene silencing (VIGS) pV190 vector as described previously (Lange et al, 2013 ; Liu et al, 2020 ). Currently, two VIGS vectors (i.e., the CGMMV‐based pV190 vector and a tobacco rattle virus [TRV]‐based vector) have been reported to be effective in gene silencing in cucurbits (Fang et al, 2021 ; Liu et al, 2020 ). In this study, we found that the pV190 vector‐induced gene silencing in watermelon fruits lasted more than 54 days and thus is suitable for studies of CGMMV infection in watermelon fruits and possibly other cucurbit crops.…”

A chitinase CsChi23 promoter polymorphism underlies cucumber resistance against Fusarium oxysporum f. sp. cucumerinum

“…To further elucidate the functions of CsChi23 and CsHB15 in Foc resistance, gene expression was knocked down in 3461 plants through a VIGS system (Fang et al ., 2021; Dong et al ., 2022). To test the suitability of the VIGS system, we conducted initial experiments using a pTRSV2 :: CsPDS construct.…”

“…For tobacco ringspot virus (TRSV)‐based VIGS experiments, we used an injection method for cucurbits (Fang et al ., 2021; Dong et al ., 2022). Briefly, the gene‐specific fragments for CsChi23 and CsHB15 were inserted into pTRSV2 vector and transformed into A. tumefaciens GV3101.…”

Identification and characterization of a novel gene involved in glandular trichome development in Nepeta tenuifolia

“…Recently, TRV has been used to determine the functions of genes associated with GT formation and development (Janga et al, 2019;Zang et al, 2021). TRV1 and TRV2 are one set of the most widely used vectors (Ratcliff et al, 2001) that have been used to silence genes in Nicotiana benthamiana, tomato, petunia, Arabidopsis, cotton, and cucumber (Liu et al, 2004;Fu et al, 2005;Fang et al, 2021). The PDS gene is a wellknown reporter used in the VIGS system, and PDS-silenced plants exhibit the photobleaching phenotypes (Liu and Page, 2008).…”