A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production

Abstract

Plant virus vectors are ideal tools for delivery of genetic cargo into host cells for functional genomics studies and protein overexpression. Although a vast number of plant virus vectors have been developed for different purposes, the utility of a particular virus vector is generally limited. Here, we report a multipurpose plant rhabdovirus-based vector system suitable for a wide range of applications in Nicotiana benthamiana. We engineered sonchus yellow net rhabdovirus (SYNV)-based gene silencing vectors through expressing a sense, antisense, or double-stranded RNAs of target genes. Robust target gene silencing was also achieved with an SYNV vector expressing a designed artificial microRNA. In addition, ectopic expression of a short tandem target mimic RNA using the SYNV vector led to a significant depletion of the target miR165/166 and caused abnormal leaf development. More importantly, SYNV was able to harbor two expression cassettes that permitted simultaneous RNA silencing and overexpression of large reporter gene. This dual capacity vector also enabled systemic expression of a whole-molecule monoclonal antibody consisting of light and heavy chains. These results highlight the utility of the SYNV vector system in gene function studies and agricultural biotechnology and provide a technical template for developing similar vectors of other economically important plant rhabdoviruses.

Keywords: RNA silencing; VIGS; antibody expression; miRNA; plant rhabdovirus; sonchus yellow net virus; viral vector.

FIGURE 1

Silencing of the GFP transgene in Nicotiana benthamiana 16c plants by SYNV VIGS vectors. (A) Schematic diagram of the genome organization of sonchus yellow net virus (SYNV) and strategies for construction of VIGS vectors. le, leader; tr, trailer; N/P J, N/P gene junction; ins., insert; int., intron; sGFP, sense GFP; asGFP, antisense GFP; hpGFP, hairpin GFP. (B) Observation of green fluorescence in plants systemically infect with SYNV VIGS vectors at 14 days after systemic infection (dpsi). Infected plants were photographed under white light (left panels) or long-wavelength UV light (middle panels). GFP fluorescence in epidermal cells of upper emerging leaves was also captured by an epifluorescence microscopy (right panels). Scale bar = 2 mm. (C) qRT PCR analysis of GFP mRNA levels in systemically infected leaf tissues at 14 dpsi. Data are means of three infected plants (n = 3). Error bars show the standard deviation (± SD). Different letters represent significant differences at p < 0.05 according to one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Western blot analysis of GFP expression in systemically infected leaf tissue at 14 dpsi. The large RuBisCO subunit (rbcL) was stained by Coomassie Brilliant Blue to indicate equal protein loading.

FIGURE 2

Silencing of N. benthamiana PDS gene by SYNV VIGS vectors. (A) Symptoms and silencing phenotypes of plants infected by SYNV vector expressing a sense (SYNV-sPDS), antisense (SYNV-asPDS), or hairpin RNA (SYNV-hpPDS) of a portion of the PDS gene at 20, 30 and 40 dpsi. (B) qRT-PCR analysis of the relative mRNA levels of PDS in systemically infected leaf tissues at 40 dpi. Data are the means of three independently infected plants (n = 3). Error bars show the standard deviation (± SD). Different letters represent significant differences at p < 0.05 according to one-way ANOVA followed by Tukey’s multiple comparisons test.

FIGURE 3

PDS gene silencing in N. benthamiana by SYNV amiRNA vector. (A) Schematic representation of amiRNA expression and the base pairing between amiRPDS and the target PDS mRNA. m⁷G: cap; A_n: poly(A) tails. miR, microRNA. (B) Phenotypes of plants infected with SYNV and SYNV-amiRPDS at 30 dpsi. (C) End-point stem-loop RT-PCR analysis of mature amiRPDS levels in systemically infected leaf tissue at 30 dpsi. The actin gene was used as an internal control. (D) qRT-PCR analysis of PDS mRNA levels in upper leaf tissues of infected plants at 30 dpsi. Data are the means of three infected plants (n = 3). Error bars show the standard deviation (± SD). Double asterisk (**) indicate significant difference at p < 0.01 according to Student’s t-test.

FIGURE 4

Suppression of miR165/166 by SYNV-STTM165/166 in N. benthamiana. (A) Schematic representation of base pairing between the STTM165/166 and miR165/166. The black arch represents the 48-nt stem-loop linker. (B) Phenotypes of plants infected with SYNV-STTM165/166 at 10 dpsi. Note: oval-shape and splitting along the edges of the blades of young emerging leaves, and downward bending of the midribs toward the stem. (C,D) Relative accumulation of miR165/166 (C) and miR165/166 target gene TC21810 (D) in systemically infected leaf tissue at 10 dpsi. Data are the means of three infected plants (n = 3). Error bars represent the standard deviation (± SD). Double asterisk (**) indicate significant difference at p < 0.01 according to Student’s t-test.

FIGURE 5

Simultaneous GUS gene expression and GFP silencing. (A) Schematic illustration of the SYNV genome organization and the strategy of GUS and hpGFP expression. The native and duplicated N/P gene junction sequences (N/P J) are depicted by the red line. The positions of the N/F and P/R primers used for RT-PCR detection of insert integrity are indicated. (B) Visualization of GFP fluorescence and GUS staining in systemically infected leaf tissues at 7 dpsi. The infected plants were photographed under white light and long-wavelength UV illumination. Upper systemically infected leaves were photographed under UV light and stained for GUS expression. (C) qRT-PCR assay to determine relative GFP mRNA levels in systemic leaves of plants infected with SYNV (EV) and SYNV-GUS-hpGFP (GUS-hpGFP) at 7 dpsi. Data are the means of three infected plants (n = 3). Error bars show the standard deviation (± SD). Double asterisk (**) indicate significant difference at p < 0.01 according to Student’s t-test. (D) Western blot analysis of GFP protein levels in systemically infected leaf tissues at 7 dpsi. The large RuBisCO subunit (rbcL) was stained with Coomassie Brilliant Blue to indicate equal protein loading. (E) RT-PCR analysis of insert stability. Total RNA was isolated from the upper infected leaves shown in (B) and was subjected to RT-PCR analysis with the N/F and P/R primers. A 1-kb DNA ladder marker is shown on the left and calculated sizes of PCR fragments are indicated on the right.

FIGURE 6

Engineering infectious SYNV vectors for expression of monoclonal antibody (mAb). (A) Schematic representation of SYNV-based dual expression vectors. LC, light chain; HC, heavy chain. The N/P gene junction sequences (N/P J) are depicted by the red line. (B) Visualization of fluorescent reporter protein expression in systemic leaves of plants infected with SYNV-GFP and SYNV-GFP-RFP at 10 dpsi. Scale bar = 500 μm. (C) Protein gel blots showing the expression of SYNV structural proteins and fluorescent reporter proteins in the leaves shown in (B). (D) ELISA detection of cucumber mosaic virus-specific mAb in SYNV-infected leaf tissue. The hybridoma-derived mAb serves as a positive control. (E) Analysis of mAbs purified from the SYNV-LC-HC_N/P leaf extract by protein gel blotting (upper panel) and Coomassie Brilliant Blue staining (lower panel). The positions of HC and LC polypeptides are indicated along with a protein size marker (M). (F) Quantification of mAb titers in N. benthamiana leaf tissues infected with SYNV vectors harboring HC and LC gene insertions. The concentrations (mg/kg leaf fresh weight) of the assembled CMV-specific mAb were determined by ELISA and quantified with a standard curve. Mock represents values in leaf extracts of SYNV-GFP infected plants. Data are the means of three individually infected plants (n = 3). Error bars show the standard deviation (± SD). Different letters represent significant differences at p < 0.05 according to one-way ANOVA followed by Tukey’s multiple comparisons test.