An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.)

Abstract

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

A circular RNA vector for targeted plant gene silencing based on an asymptomatic viroid

“…It is worth noting that significant silencing of PDS and Chll transcripts is not always accompanied by homogenous phenotypic alterations in systemically infected eggplant leaves (Figures 2 and 4 ). This observation is consistent with previous results showing that VIGS by short PDS ‐insertions (below 120 nt) triggered minimum or no bleaching phenotype in plants infected with modified brome mosaic virus (Wang et al., 2021 ), or barley stripe mosaic virus (Bruun‐Rasmussen et al., 2007 ), although measurements of the respective transcripts levels showed significant reductions. In one of these cases (Bruun‐Rasmussen et al., 2007 ), it was suggested that to trigger a visible bleaching phenotype PDS transcripts levels should be lower than a specific threshold at a certain point in leaf development.…”

“…Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 112, 284-293 in plants infected with modified brome virus (Wang et al, 2021), or barley stripe mosaic virus (Bruun-Rasmussen et al, 2007), although measurements of the respective transcripts levels showed significant reductions. In one of these cases (Bruun-Rasmussen et al, 2007), it was suggested that to trigger a visible bleaching phenotype PDS transcripts levels should be lower than a specific threshold at a certain point in leaf development.…”

“…However, the observation that plants infected with ELVd‐42 had an approximately 90% reduction in PDS mRNA accumulation and exhibited irregular bleaching (Figure 2 ) indicates that this threshold was reached. To better understand the patchiness, it may be interesting to test for the presence of the insert and the target gene silencing in different portions of leaves showing patches of visible silencing to help explain these phenotypic inconsistencies (Wang et al., 2021 ).…”

Foxtail mosaic virus-induced gene silencing (VIGS) in switchgrass (Panicum virgatum L.)

“…Using this approach, systemic infection was achieved in approximately one quarter (~ 28%) of the tested 3- or 8-week-old switchgrass seedlings. This infection efficiency is consistent with other monocot species, where plant susceptibility to viral infection has been shown to be impacted by other factors such as genotype/cultivar, vector chassis, construct design, and growth conditions [ 48 , 49 ]. The relevance of optimizing construct design is exemplified by the instability of the FoMV:PDS construct observed in this study.…”

“…Although CRISPR/Cas9 and RNAi protocols have been successfully employed in switchgrass [ 8 , 10 , 11 , 14 , 46 , 47 ] , the complex allotetraploid genome and largely outcrossing nature of switchgrass complicate genetic studies. In this study, we used the FoMV vector and mechanical rub-inoculation, previously established in maize, sorghum, and wheat [ 26 , 48 , 49 ] to achieve VIGS in switchgrass. Using this approach, systemic infection was achieved in approximately one quarter (~ 28%) of the tested 3- or 8-week-old switchgrass seedlings.…”