Foxtail mosaic virus: A Viral Vector for Protein Expression in Cereals

Abstract

Rapid and cost-effective virus-derived transient expression systems for plants are invaluable in elucidating gene function and are particularly useful in plant species for which transformation-based methods are unavailable or are too time and labor demanding, such as wheat (Triticum aestivum) and maize (Zea mays). The virus-mediated overexpression (VOX) vectors based on Barley stripe mosaic virus and Wheat streak mosaic virus described previously for these species are incapable of expressing free recombinant proteins of more than 150 to 250 amino acids, are not suited for high-throughput screens, and have other limitations. In this study, we report the development of a VOX vector based on a monopartite single-stranded positive sense RNA virus, Foxtail mosaic virus (genus Potexvirus). In this vector, PV101, the gene of interest was inserted downstream of the duplicated subgenomic promoter of the viral coat protein gene, and the corresponding protein was expressed in its free form. The vector allowed the expression of a 239-amino acid-long GFP in both virus-inoculated and upper uninoculated (systemic) leaves of wheat and maize and directed the systemic expression of a larger approximately 600-amino acid protein, GUSPlus, in maize. Moreover, we demonstrated that PV101 can be used for in planta expression and functional analysis of apoplastic pathogen effector proteins such as the host-specific toxin ToxA of Parastagonospora nodorum Therefore, this VOX vector opens possibilities for functional genomics studies in two important cereal crops.

Figure 1.

Testing the infectivity of the first-generation FoMV vector pGR-FoMV.pCF in N. benthamiana and wheat. A, Upper uninoculated leaves from mock- or virus-inoculated N. benthamiana plants at 19 dpi. Bars = 20 mm. B, Upper uninoculated leaves from mock- or virus-inoculated wheat cv Riband plants at 13 dpi. Bars = 20 mm. C and D, Detection of FoMV RNA in upper uninoculated leaves from mock- or virus-inoculated N. benthamiana (C) and wheat (D) plants using RT-PCR. Housekeeping N. benthamiana Protein phosphatase 2 (PP2A; C) and wheat Cell division control 48 (CDC48; D) genes were used as loading controls.

Figure 2.

Development of a FoMV expression vector. A, FoMV genome organization and expression strategy. The viral genomic RNA (gRNA) contains five major ORFs (labeled from 1 to 4 and CP), coding for the polymerase (ORF1), movement proteins (ORF2–ORF4), and CP, and a cryptic ORF5A that gives rise to an N-terminal CP extension with unknown function. ORF1 is expressed from gRNA, whereas ORF2, ORF3, and ORF4 and CP are expressed from sgRNA1 and sgRNA2, respectively, which are synthesized by the viral polymerase. The synthesis of sgRNAs is driven by subgenomic promoters sgp1 and sgp2. Black circle, mRNA cap structure; A_n, poly(A) tail; black arrow, sgRNA transcription start. B, A series of FoMV expression vectors were constructed by duplicating differently sized predicted sgp2 sequences, each encompassing a conserved eight-nucleotide core element (core). Duplicated sequences were placed downstream of the ORF5A start codon, disrupting the synthesis of an N-terminal CP extension. C, Schematic diagram of the constructed FoMV expression vectors. The gene of interest (GOI) in these vectors is inserted between sgp2.1 and sgp2 by restriction enzyme (isolate pCF-based vectors and PV101 based on the isolate PV139) or Gateway cloning (PV101gw) and expressed from an additional sgRNA2.1 generated from sgp2.1. The spacing between sgp2.1 and sgp2 is drawn not to scale. 35S, Cauliflower mosaic virus (CaMV) 35S promoter; nos, nopaline synthase terminator; *, start codon of ORF5A.

Figure 3.

Expression of GFP from the first-generation FoMV VOX vectors in N. benthamiana. A series of four expression vectors generated through the duplication of 45-, 55-, 90-, and 101-nucleotide sequences spanning the predicted sgp2 sequences were generated based on the full-length infectious FoMV cDNA clone pCF. A, N. benthamiana leaves coinfiltrated with one of the A. tumefaciens strains carrying either the empty vector pCF101 or pCF101-GFP, pCF90-GFP, pCF55-GFP, or pCF45-GFP and an A. tumefaciens strain carrying a construct for the expression of the gene-silencing suppressor p19. Representative infiltrated leaves were photographed at 6 dpi with a fluorescence stereomicroscope mounted with band-pass (BP) and long-pass (LP) filters using identical acquisition settings. Bars = 2.5 mm. B, Immunodetection of FoMV CP and GFP in pooled agroinfiltrated leaves from three individuals sampled at 3 dpi using the corresponding antibodies. Equal loading was verified by staining the membranes with Ponceau S.

Figure 4.

Influence of growth conditions on FoMV-mediated protein expression. A, Seedlings of wheat cv Riband grown under standard or optimized growth conditions were inoculated with pCF101-GFP. Mock-inoculated plants or plants inoculated with the wild-type FoMV pCF served as negative controls. Inoculated leaves (L2) from three representative individual plants were photographed at 8 dpi using a fluorescence stereomicroscope mounted with band-pass (BP) and long-pass (LP) filters. B, Scant green fluorescent foci observed in the upper uninoculated leaves (L4) of some pCF101-GFP-inoculated plants grown under optimized conditions at 18 dpi. All fluorescence images in A and B were taken using identical acquisition settings. Bars = 2.5 mm.

Figure 5.

Generation of a full-length infectious cDNA clone of the FoMV isolate PV139. A and B, Symptoms observed on the upper uninoculated leaves of wheat cv Riband (A) and Bobwhite (B) plants infected with the original FoMV PV139 at 21 dpi. No symptoms were observed in mock-inoculated plants. Bars = 20 mm. C, Pipeline used to obtain a consensus master genome sequence of FoMV isolate PV139 starting from a small RNA fraction purified from the FoMV-infected leaf material. INDEL, Insertion/deletion polymorphism; SNP, single-nucleotide polymorphism. D, Symptoms observed on the upper uninoculated leaves of wheat cv Riband plants infected with the full-length infectious FoMV PV139 cDNA clone at 24 dpi. No symptoms were observed in mock-inoculated plants. Bars = 20 mm. E, Detection of FoMV RNA in the upper uninoculated leaves of wheat cv Riband plants infected with the FoMV PV139 cDNA clone by RT-PCR. Wheat CDC48 was used as a loading control.

Figure 6.

Expression of GFP using the second-generation FoMV vector PV101 in plants. A, Directly inoculated (via coinfiltration with A. tumefaciens strains carrying PV101-GFP and a construct for the expression of the gene-silencing suppressor p19) and upper uninoculated leaves of N. benthamiana plants at 7 and 14 dpi, respectively. Bars = 20 mm. B, Directly inoculated and upper uninoculated (systemic) leaves of wheat cv Riband at 7 and 14 dpi, respectively. Bars = 2.5 mm. C, Systemically infected leaves of maize line B73 plants at 20 dpi. Bar = 50 mm. Photographs were taken using a camera (A and C) or a fluorescence stereomicroscope (B) mounted with band-pass (BP) and long-pass (LP) filters. D, Immunodetection of GFP and FoMV-CP in pooled systemically infected leaves from three individuals of different plant species sampled at 14 dpi using the corresponding antibodies. The presence of fluorescence in PV101-GFP-infected plants was checked before sampling. Equal loading was verified by staining the membranes with Ponceau S.

Figure 7.

Influence of the wheat genotype on FoMV-mediated protein expression. PV101-GFP-inoculated leaves (L2) of different wheat cultivars were photographed at 8 dpi using a fluorescence stereomicroscope mounted with band-pass (BP) and long-pass (LP) filters. All fluorescence images were taken using identical acquisition settings. The maximum scores for GFP fluorescence coverage in systemically infected leaves of wheat cv Riband (Rib), Bobwhite (BW), Chinese Spring (CS), Halberd (Hal), Grandin (Grd), Sumai 3 (Su3), Cadenza (Cad), Paragon (Par), and Pakito (Pak) are indicated. Data are from at least 16 plants from three independent experiments. Bars = 2.5 mm.

Figure 8.

Expression of the 600-amino acid-long protein GUSPlus using the second-generation FoMV vector PV101. PV101-GUSPlus was inoculated onto wheat cv Pakito (A) and Riband (B) and onto maize line B73 (C), and GUSPlus activity in leaf samples from inoculated plants was detected by histochemical staining with 5-bromo-4-chloro-3-indolyl-β-d-GlcA. An empty vector, PV101, was used as a control. Samples of inoculated leaves were taken at 9 dpi. Samples of first systemic wheat (L3) and maize (L4) leaves were taken at 15 dpi, and samples of second systemic wheat leaves (L4) and second and third maize leaves (L5 and L6) were taken at 22 dpi. Each leaf piece comes from a different individual plant. PV101- and PV101GUSPlus-infected material was sampled from four representative individuals from two independent experiments and from six representative individuals from three independent experiments, respectively. Bars = 20 mm.

Figure 9.

FoMV-mediated expression of the necrotrophic fungal effector ToxA from P. nodorum. A, PV101-GFP- or PV101-ToxA-inoculated leaves (L2) of wheat cv Chinese Spring (CS; ToxA insensitive) and Halberd (Hal; ToxA sensitive) seedlings at 6 dpi from one of the two replicated experiments. Bar = 20 mm. B, Upper uninoculated leaves (L3) from wheat cv Chinese Spring and Halberd plants inoculated with PV101 carrying either full-length SnToxA effector protein with its native secretion signal peptide or its mature version without signal peptide (ToxA_noSP) at 11 dpi (and 16 dpi where indicated). Photographs were taken from the same leaf areas under white light or blue light using a fluorescence stereomicroscope mounted with a long-pass filter (LP). Bars = 2.5 mm.