Abstract
Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) has been frequently used in dicots. Here we show that it can also be used in monocots, by presenting a system involving use of a novel infiltration solution (containing acetosyringone, cysteine, and Tween 20) that enables whole-plant level VIGS of (germinated) seeds in wheat and maize. Using the established system, phytoene desaturase (PDS) genes were successfully silenced, resulting in typical photo-bleaching symptoms in the leaves of treated wheat and maize. In addition, three wheat homoeoalleles of MLO, a key gene repressing defense responses to powdery mildew in wheat, were simultaneously silenced in susceptible wheat with this system, resulting in it becoming resistant to powdery mildew. The system has the advantages generally associated with TRV-mediated VIGS systems (e.g., high-efficiency, mild virus infection symptoms, and effectiveness in different organs). However, it also has the following further advantages: (germinated) seed-stage agroinfiltration; greater rapidity and convenience; whole-plant level gene silencing; adequately stable transformation; and suitability for studying functions of genes involved in seed germination and early plant development stages.
Highlights
virus-induced gene silencing (VIGS) tools for maize is lacking, except the VIGS vectors derived from brome mosaic virus (BMV) (Ding et al, 2006) and recently reported virus vectors based on cucumber mosaic virus (CMV) strain ZMBJ-CMV and foxtail mosaic virus (FoMV) (Liu et al, 2016; Mei et al, 2016; Wang R. et al, 2016), the photo-bleaching symptom by silencing phytoene desaturase (PDS) gene is partial, suggesting that those VIGS systems in maize could not function at wholeplant level
After the seeds had been transferred to soil and cultivated in growth chambers for 16 days, systemic photo-bleaching was observed in all leaves of plants that developed from pTRV-SlPDS-inoculated germinated wheat seeds (Figures 1F–H)
Partial photo-bleaching was observed in leaves of plants that developed from wheat seeds that were inoculated with pTRV-SlPDS before germination (Figures 1J–L), and control plants infected by pTRV2 developed normal leaves with no photo-bleaching symptoms under the same growth conditions (Figures 1C–E) These findings clearly show that TaPDS was silenced in pTRV-SlPDS infected plants. quantitative RT-PCR (qRT-PCR) performed to check whether PDS was silenced in the whole plants, qRTPCR data confirmed that PDS expression was knocked down in both roots and leaves of pTRV-SlPDS plants (Supplementary Figure S4)
Summary
With fast development and wide applications of next-generation sequencing (NGS) technologies, genomes of increasing numbers of plants have been sequenced, including important crops such as barley, wheat, maize, cotton, and tomato (Schnable et al, 2009; Bräutigam and Gowik, 2010; Zhou et al, 2010; Brenchley et al, 2012; Kumar et al, 2012; Liu et al, 2012; Mayer et al, 2012; Paterson et al, 2012; The Tomato Genome Consortium, 2012; Jia et al, 2013; Ling et al, 2013). A widely used approach in functional analysis is to generate mutations using chemical or physical agents (Kodym and Afza, 2003; Belfield et al, 2012; Hanafy and Mohamed, 2014; Serrat et al, 2014; Dhaliwal et al, 2015; Li et al, 2016; Zhang et al, 2016), T-DNA insertion (Weigel et al, 2000; Alonso et al, 2003), RNA interference (RNAi) (Kusaba, 2004; Mahmood-ur-Rahman et al, 2008; Wang et al, 2008, 2013), or genome editing (Shan et al, 2013; Wang et al, 2014; Kumar and Jain, 2015) Those techniques often generate large numbers of mutants, and require tedious screening or genetic transformation procedures. VIGS tools for maize is lacking, except the VIGS vectors derived from brome mosaic virus (BMV) (Ding et al, 2006) and recently reported virus vectors based on cucumber mosaic virus (CMV) strain ZMBJ-CMV and foxtail mosaic virus (FoMV) (Liu et al, 2016; Mei et al, 2016; Wang R. et al, 2016), the photo-bleaching symptom by silencing phytoene desaturase (PDS) gene is partial, suggesting that those VIGS systems in maize could not function at wholeplant level
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