A Virus-Induced Assay for Functional Dissection and Analysis of Monocot and Dicot Flowering Time Genes

Abstract

Virus-induced flowering (VIF) uses virus vectors to express Flowering Locus T (FT) to induce flowering in plants. This approach has recently attracted wide interest for its practical applications in accelerating breeding in crops and woody fruit trees. However, the insight into VIF and its potential as a powerful tool for dissecting florigenic proteins remained to be elucidated. Here, we describe the mechanism and further applications of Potato virus X (PVX)-based VIF in the short-day Nicotiana tabacum cultivar Maryland Mammoth. Ectopic delivery of Arabidopsis (Arabidopsis thaliana) AtFT by PVX/AtFT did not induce the expression of the endogenous FT ortholog NtFT4; however, it was sufficient to trigger flowering in Maryland Mammoth plants grown under noninductive long-day conditions. Infected tobacco plants developed no systemic symptoms, and the PVX-based VIF did not cause transgenerational flowering. We showed that the PVX-based VIF is a much more rapid method to examine the impacts of single amino acid mutations on AtFT for floral induction than making individual transgenic Arabidopsis lines for each mutation. We also used the PVX-based VIF to demonstrate that adding a His-or FLAG-tag to the N or C terminus of AtFT could affect its florigenic activity and that this system can be applied to assay the function of FT genes from heterologous species, including tomato (Solanum lycopersicum) SFT and rice (Oryza sativa) Hd3a. Thus, the PVX-based VIF represents a simple and efficient system to identify individual amino acids that are essential for FT-mediated floral induction and to test the ability of mono-and dicotyledonous FT genes and FT fusion proteins to induce flowering.Modified plant viruses have emerged as powerful tools for dissecting gene function in plants. Such plant virus-based technology can be applied to facilitate or impede gene expression, resulting in gain-or loss-offunction phenotypes. Although virus-based technology was initially exploited for the purpose of high-level production of foreign proteins, such as recombinant subunit vaccines and pharmaceutical proteins for molecular pharming (Scholthof et al., 1996;Porta and Lomonossoff, 2002), plant RNA and DNA virus-based techniques such as small interfering RNA-mediated virus-induced posttranscriptional gene silencing (VIGS) has been extensively utilized to silence genes for functional genomic studies in dicots and monocots, including plants and crops recalcitrant to classical forward or reverse genetic manipulation (Lindbo et al

An RNAi suppressor activates in planta virus–mediated gene editing

“…Transgenic plant-free transient toolboxes such as virus induced gene silencing (VIGS; Lin et al, 2008;Chen et al, 2015), gene complementation (VIGC; Zhou et al, 2012) and flowering (VIF; Li et al, 2011;Qin et al, 2017) have been widely used in plants. To date, a bona fide non-transgenic virus-mediated GE (VmGE) platform has not been established to induce inheritable gene modifications although several RNA/DNA viruses were utilized to express sgRNA for GE in Cas9-transgenic plants (Ali et al, 2015;Yin et al, 2015;Cody et al, 2017).…”

Overexpression of Lilium formosanum MADS-box (LFMADS) Causing Floral Defects While Promoting Flowering in Arabidopsis thaliana, Whereas Only Affecting Floral Transition Time in Nicotiana tabacum

“…In addition, the effect of the overexpressed LFMADS2 , LFMADS4 , and LFMADS6 on the transcript levels of regulatory genes related to the flowering time in tobacco was examined. Relative transcript levels of the flowering-related genes, including NFL2 , NtCO , NtFT1 , NtFT2 , NtFT3 , NtFT4 , NtFUL I , and NtSOC1 [ 73 , 74 , 75 ] were determined by real-time qPCR analyses of WT and transgenic tobaccos. Our real-time qRT-PCR analysis indicated that LFMADS2 , LFMADS4 , and LFMADS6 significantly expressed integrator genes, NtFUL and NtSOC1 , which may affect floral transition causing early flowering in transgenic tobaccos ( Figure 10 and Figure 11 A).…”

Virus-induced overexpression of heterologous FT for efficient speed breeding in tomato

“…This vector is infectious on a broad range of Solanaceous plants, was initially developed for VOX (Chapman et al 1992) but has been used widely for virus-induced gene silencing (VIGS) (Lu et al 2003;Ruiz et al 1998). PVX-based VOX of FT in tobacco was used in a mutation analysis of FT function (Qin et al 2017) but, previously it had not been tested as system for speed breeding of tomato.…”