Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

Abstract

Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops.

Figure 1.

Schematic organization of FoMV genome and infectious FoMV vector. A, Genomic organization of FoMV RNA. The ORFs are indicated by boxes. The 152-kD protein is an RNA-dependent RNA polymerase. The three proteins at 26 kD, 11.3 kD, and 5.8 kD, collectively known as a triple gene block, are presumed to mediate viral cell-to-cell moment. The 5A protein was produced in vivo; the 24-kD CP is located at the 3′-terminus. B, Schematic of infectious FoMV T-DNA clone pFoMV-wt. FoMV was cloned between CaMV 35S promoter with duplicated enhancers and a nopaline synthase (NOS) terminator of a T-DNA binary vector. pFoMV-wt has an additional 70 adenosine residues after the viral 3′-terminus. C, Schematic of the FoMV VIGS vector, pFoMV-sg. pFoMV-sg is a pFoMV-wt derivative that contains a direct repeat of the 170-bp putative FoMV CP subgenomic promoter (GenBank accession: EF630359, nt 5202–5371), shown by arrows, and multiple cloning sites (HpaI, MluI, XhoI, and AscI) between CP subgenomic promoters. Right- and left-pointing blue arrows indicate sense and anti-sense orientation of foreign fragments, and the predicted structure of RNA transcript is presented on the right side. D, Symptom development and RT-PCR detection of viral RNA showed that pFoMV-wt and pFoMV-sg are infectious in barley. Bar = 1 cm.

Figure 2.

FoMV-based VIGS of PDS and ChlH in barley. A, Barley plants were infected with a pFoMV-sg vector carrying an inverted-repeat fragment of HvPDS (FoMV-HvPDS) and HvChlH (FoMV-HvChlH). The third uninoculated leaves were photographed at 21 dpi. Bar = 1 cm. B, Quantification of the mRNA levels of HvPDS and HvChlH of barley plants was analyzed by real-time RT-PCR, normalized relative to the level of 18S rRNA. ***P < 0.01. Measurements were repeated three times, respectively. The error bar indicates se.

Figure 3.

FoMV-based VIGS of marker genes in foxtail millet. Foxtail millet plants were infected with a pFoMV-sg or pFoMV-sg vector carrying inverted-repeat fragments of SiPDS (FoMV-SiPDS), SiCLA1 (FoMV-SiCLA1), and SiIspH (FoMV-SiIspH), respectively. Photographs were taken for the seventh leaf (7L) at 24 dpi (A), for the 10th leaf (10L) at 39 dpi (C), and for the 13th leaf (13L) at 55 dpi (E). Bars = 1 cm. Quantification of mRNA levels of three marker genes of foxtail millet plants at 24 dpi (B), 39 dpi (D), and 55 dpi (F) were analyzed by real-time RT-PCR, normalized relative to the value of 18S rRNA. ***P < 0.01. Measurements were repeated three times, respectively. The error bar indicates se.

Figure 4.

FoMV-based VIGS of PDS and CLA1 in wheat. A, Wheat plants were infected with a pFoMV-sg vector carrying an inverted-repeat fragment of TaPDS (FoMV-TaPDS) or TaCLA1 (FoMV-TaCLA1). The fourth leaves were photographed at 35 dpi. Bar = 1 cm. B, Quantification of the mRNA levels of TaPDS and TaCLA1 of wheat plants was analyzed by real-time RT-PCR, normalized relative to the level of 18S rRNA. **P < 0.05; ***P < 0.01. Measurements were repeated three times, respectively. The error bar indicates se.