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Development of new potato virus X-based vectors for gene over-expression and gene silencing assay
Virus Research 191 (2014) 62–69
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Development of new potato virus X-based vectors for gene over-expression and gene silencing assay Ying Wang a,b,1 , Qian-Qian Cong a,1 , Yu-Fei Lan c,1 , Chao Geng a , Xian-Dao Li a , Yuan-Cun Liang a , Zheng-You Yang b,∗∗ , Xiao-Ping Zhu a , Xiang-Dong Li a,∗ a
Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Shandong 271018, China Department of Microbiology, College of Life Sciences, Shandong Agricultural University, Shandong 271018, China c Tai’an Academy of Agricultural Sciences, Shandong 271000, China b
a r t i c l e
i n f o
Article history: Received 16 April 2014 Received in revised form 16 July 2014 Accepted 20 July 2014 Available online 27 July 2014 Keywords: Potato virus X (PVX) Over-expression Gene silencing Gene vector Ligation independent cloning
a b s t r a c t Multiple plant viruses, including potato virus X (PVX), have been modified as vectors for expressing heterologous genes or silencing endogenous genes in plants. PVX-based vectors facilitate the functional analysis of genes in plant. However, they can only express one protein in a time. In this paper we report the construction of new vectors based on a 35S promoter-driven PVX infectious clone, pCaPVX100. Vector pCaPVX440 contains two additional subgenomic promoters and can be utilized to express two foreign genes at the same time. Plasmid pCaPVX760 is a CP minus vector and can be used to express foreign proteins through the gene substitution strategy. In addition, plasmid pCaPVX100 was engineered into a gene silencing vector (pCaPVX440-LIC) by introducing a ligation independent cloning (LIC) site into the vector. These results indicate that the newly developed PVX vectors are competent for multiple research purposes. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years multiple plant RNA and DNA viruses have been modified to serve as vectors for over-expressing and/or silencing genes in plants. Some virus-based vectors were used to produce heterologous proteins or peptides with commercial importance, including antibodies or vaccine antigens, in plant cells (Gellért et al., 2012; Gleba et al., 2007; Nuzzaci et al., 2007; Roy et al., 2010). In general, plant virus-based over-expression systems are more cost-effective and easier to use than the stable transformation technology. The virus-based vectors can also be used to study gene function via virus-induced gene silencing (VIGS) (BurchSmith et al., 2004; Faivre-Rampant et al., 2004; Kumar et al., 2012; Purkayastha et al., 2010; Zhang et al., 2009, 2012). Silencing genes in plant often results in specific phenotypes that allow quick identifications of gene functions in plants. To date more than a dozen plant virus-based vectors have been used successfully to silence genes in plants, including apple latent spherical virus (Igarashi et al.,
∗ Corresponding author. Tel.: +86 (0) 538 8242523; fax: +86 (0) 538 8226399. ∗∗ Corresponding author. E-mail addresses: [email protected] (Z.-Y. Yang), [email protected], [email protected] (X.-D. Li). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.virusres.2014.07.018 0168-1702/© 2014 Elsevier B.V. All rights reserved.
2009), barley stripe mosaic virus (Yuan et al., 2011), bean pod mottle virus (BPMV) (Zhang et al., 2009, 2010), brome mosaic virus (Ding et al., 2006), cabbage leaf curl virus (Turnage et al., 2002), cotton leaf crumple virus (Tuttle et al., 2008), grapevine leafrollassociated virus-2 (Kurth et al., 2012), pea early browning virus (Constantin et al., 2004), potato virus X (PVX) (Faivre-Rampant et al., 2004; Lacomme and Chapman, 2008), tobacco mosaic virus (TMV) (Kumagai et al., 1995), tobacco rattle virus (Burch-Smith et al., 2006; Kumar et al., 2012; Ratcliff et al., 2001; Valentine et al., 2004), and tomato golden mosaic virus (Peele et al., 2001). PVX has a single-stranded genomic RNA of about 6430 nucleotides (nt) in length (Yu et al., 2010). The 5 end of the PVX RNA contains an m7 GpppG cap structure and its 3 end has a polyadenylate tail. The PVX genome has five open reading frames (ORFs). The ORF1 encodes a 164 kDa RNA-dependent RNA polymerase (RdRp) followed by three partially overlapped ORFs known as the triple gene block (TGB). The TGB encodes the 25 kDa TGBp1, the 12 kDa TGBp2, and the 8 kDa TGBp3, respectively. The last ORF in the PVX genome, ORF5, is also transcribed through a subgenomic promoter (SGP) and encodes the 25 kDa structural coat protein (CP). The TGBps and CP have been shown to function in PVX cell-to cell movement in plants (Fedorkin et al., 2001; Lough et al., 2000). PVX genome was cloned behind a T7 RNA polymerase promoter or a cauliflower mosaic virus (CaMV) 35S promoter to serve as expression and/or gene silencing vectors (Lacomme and Chapman,
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2008). The PVX-based vectors have been successfully used to transiently express foreign genes; however, they can only express one protein in a time (Cerovska et al., 2012; Chapman et al., 1992; Lacomme and Chapman, 2008; Plchova et al., 2011). PVX-based vectors have also been used to silence endogenous genes through VIGS, so as to study the function of silenced gene (Faivre-Rampant et al., 2004; Lacomme and Chapman, 2008). However, digestion and ligation procedures limit its application in large scale cloning. In this paper, we constructed new PVX-based expression vectors that can express two foreign genes simultaneously in the same cells, or express foreign proteins through the gene substitution strategy. In addition, a VIGS vector was constructed, which carries a ligation independent cloning (LIC) site and can be used in high throughout silencing studies. 2. Materials and methods 2.1. Growth of test plants Seeds of Nicotiana benthamiana, tobacco (N. tabacum) cv. NC89, tomato (Solanum lycopersicum) cv. Micro-Tom and potato (S. tuberosum) cv. Zaodabai were sown in soil. The seedlings were transplanted into pots at the 3- to 4-leaf stage, and allowed to grow in a growth chamber set at 25 ◦ C and 16 h/8 h (light/dark) conditions. 2.2. Construction of PVX infectious clone The PVX isolate 1985 (PVX1985; GenBank accession number EU571480) was from a previously published source (Yu et al., 2010) and maintained in N. tabacum plants. The PVX1985 sequence was amplified through reverse transcription (RT)-PCR from a total RNA sample extracted from infected N. tabacum leaves using a moloney murine leukemia virus (M-MuLV) reverse transcriptase (Transgen, China) and a Phusion DNA Polymerase (Thermo, Finland). To construct the full-length infectious clone of PVX1985, a 35S promoter and the 5 end 548 bp sequence of PVX genome were fused together through overlapping PCR with primer pairs 1985pro-F (‘F’ for forward) and 1985-pro-R (‘R’ for reverse), 1985-1-F and 1985-1-R (Table S1), and then ligated into vector pMD18-T simple to yield pMD18-T-1. The second fragment representing partial PVX sequence, position 543 to 3354, was amplified with the primer pair 1985-2-F and 1985-2-R. After digestion with EcoRI and XmaI enzymes, the fragment was ligated into pMD18-T-1 to yield pMD18-T-2. The 3 half PVX genome, position 3349 to the 3 -end poly (A) tail, was amplified with the primer pair 1985-3-F and 1985-3-R, digested with BamHI and XmaI, and then ligated into pMD18-T-2 to yield pMD18-T-PVX100. The full-length 35S promoter and the PVX1985 sequence were then inserted between the SalI and XmaI site in pCambia0390. The resulting vector is referred to as pCaPVX100 (Fig. 1A). The PVX100 nucleotide sequence is numbered as the PVX sequence deposited in GenBank. 2.3. Construction of PVX-based over-expression and silencing vectors To construct a pCaPVX100-based over-expression vector, we planned to introduce a multiple clone site to the region between TGB and CP ORF. However, the candidate restriction site of ApaI was also present in the plasmid pCambia0390. Therefore, we first obtained an insert containing three fragments. The first fragment contained the sequence from ApaI site (4945) to PVX CP SGP (Chapman et al., 1992; Skryabin et al., 1988) plus an AsiSI restriction site (GCG ATC GC). The second fragment contained the TMV CP SGP (Grdzelishvili et al., 2000) fused with the second cloning site (e.g. SacI, BstBI and MluI; GAG CTCGGT CCG GAG GTT CGA ACG
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ACG CGT). The third fragment contained the 3 -terminal sequence of PVX1985 genome from the PVX CP SGP to the 3 -end poly (A) tail. These three fragments were amplified separately with the primer pairs 1985-4-F/1985-7-R, 1985-6-F/1985-6-R, and 19857-F/1985-3-R, and fused by overlapping PCR with primer pair 1985-4-F and 1985-3-R. The insert was digested with ApaI and XmaI and cloned into pMD18-T-PVX100 to produce pMD18-T-PVX140. The 35S promoter sequence and the engineered PVX genome in pMD18-T-PVX140 were then cloned into pCambia0390 using the restriction sites SalI and XmaI. The thymine in the first PVX CP SGP (position 5651) and TMV CP SGP (position 5859) were changed to guanine through mutagenesis PCR to abolish the start codon. The resultant plasmid was designated as pCaPVX440 (Fig. 2A). The second over-expression vector pCaPVX760 was produced by replacing most of the PVX CP sequence (ORF5), position 6009 to 6,710, in the pCaPVX440 with three restriction sites (NheI, XhoI and NruI; GCT AGCAAA CTC GAGTTT TCG CGA). The three NheI sites in pCaPVX440 were removed by changing GCT AGC to GCA AGC (Fig. 3A). The fragments encoding the N- or C-terminal parts of an enhanced yellow fluorescent protein (eYFP) were amplified from vectors 35S-SPYNE and 35S-SPYCE (Walter et al., 2004), respectively, using the primer pairs YFP-Sac-F/Lap-YFP-R and LapYFP-F/YFP-Mlu-R (Table S1), and then used to generate the full length eyfp gene via overlapping PCR. The green fluorescent protein gene (gfp) was amplified from pTVBMV-GFP (Gao et al., 2012) through PCR using the primer pairs GFP-Asis-F/GFP-Asis-R and GFP-Nhe-F/GFP-Nru-R. The resultant gfp and eyfp fragments were inserted individually into vectors pCaPVX440 and pCaPVX760 to generate pCaPVX440-GFP, pCaPVX440-GFP-YFP and pCaPVX760GFP. Vector pCaPVX440-LIC (Fig. 4A) was produced by inserting a LIC sequence (AGG GTC TTG TCG TTC GAA CCC GAG AGG AGT A) (Aslanidis and de Jong, 1990; Dong et al., 2007; Yuan et al., 2011) containing a BstBI restriction site (underlined sequence) between the SacI and MluI sites in pCaPVX440. The pds gene was amplified with primer pairs Lic-PDS-F/Lic-PDS-R and LicPDSas-F/Lic-PDSas-R, in which the forward primers contained 5 -GGGTCTTGTCGTTCGA-3 and the reverse primers contained 5 CTCCTCTCGGGTTCGA-3 at their 5 -termini. By using primer pairs Lic-PDS-F/Lic-PDS-R and Lic-PDSas-F/Lic-PDSas-R, the same region of the pds gene was amplified, but ligated into the silencing vector pCaPVX440-LIC in different orientations. The 717 bp fragment of gfp gene was amplified with primers Lic-GFP-F and Lic-GFP-R. The PCR products were purified, and then treated at 22 ◦ C for 30 min with T4 DNA polymerase (New England Biolabs) in 1X reaction buffer containing 5 mM dTTP to generate sticky ends, and heated at 75 ◦ C for 10 min to inactivate the polymerase. The BstBI-linearized pCaPVX440-LIC vector was treated with T4 DNA polymerase in the presence of 5 mM dATP to generate sticky ends complementary to those of the PCR products mentioned above. The treated PCR products (∼200 ng) and pCaPVX440-LIC vector (∼20 ng) were mixed, incubated at 66 ◦ C for 2 min and 22 ◦ C for 10–30 min to anneal their complementary termini. Then 10 L aliquots were used for transformation into Escherichia coli DH5␣. Positive clones identified by plasmid PCR and restriction enzyme digestion were confirmed by sequencing. 2.4. Detection of the pds gene The pds gene accumulation level in N. benthamiana was detected at 14 dpi by semi-quantitative RT-PCR using primers PDS-1-F and PDS-1-R, which were complementary to regions outside the one cloned into pCaPVX440-LIC in 2.3. The semi-quantitative RT-PCR reaction was conducted in a final volume of 20 L, containing 250 ng of RNA and 0.5 M of each primer. After 27 cycles, the PCR
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Fig. 1. Analysis of pCaPVX100 infectivity in different host plants. (A) Schematic representation of pCaPVX100 and restriction sites used for cloning. 35S, the 35S promoter of CaMV; RdRp, RNA-dependent RNA polymerase; 25 kDa, 12 kDa and 8 kDa represent the proteins encoded by the triple gene block (TGB); CP, coat protein. Black arrow indicates the position of PVX CP subgenomic promoter. (B) Symptoms caused by the wild type isolate PVX1985 and the infectious clone pCaPVX100 in N. benthamiana, tobacco (N. tabacum) cv. NC89, potato (Solanum tuberosum) cv. Zaodabai and tomato (S. lycopersicum) cv. Micro-Tom at 5, 11 or 15 days post inoculation. (C) Detection of PVX CP RNA accumulation in different host plants through RT-PCR. Mo, mock inoculation; Mi, mechanical inoculation of PVX1985; Ai, agro-infiltration of pCaPVX100; M, DNA ladder.
products were analyzed in 1% agarose gels. The EF1␣ mRNA was used as an internal RNA control. 2.5. Inoculation and fluorescence visualization Plasmid pCaPVX100 and its derivatives were transformed individually into Agrobacterium tumefaciens GV3101 cells following the freeze and thaw transformation procedure (Cui et al., 1995). Overnight grown Agrobacterium cultures were pelleted through centrifugation at 5,000 × g for 3 min and then incubated in an
induction solution (10 mmol/L MES, pH 5.8, 0.1 mmol/L acetosyringone and 10 mmol/L MgCl2 ) for 3 h. Leaves of assay plants were infiltrated with individual Agrobacterium culture (OD600 = 0.5) using needle-less syringes. Leaves infiltrated with Agrobacterium harboring gfp gene-carrying constructs were examined for GFP fluorescence using a hand-held UV (365 nm wavelength) lamp (Blak Ray B100-AP lamp, UV products, Upland, CA 91786, USA). Every experiment was repeated three times independently. Three to five plants were agroinoculated by each plasmid every time.
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Fig. 2. Genomic structure of vector pCaPVX440 and its application in over-expression. (A) Schematic representation of the over-expression vector pCaPVX440. Black arrows indicate the PVX CP subgenomic promoters, green arrow indicates the position of TMV CP subgenomic promoter. (B) Over-expression of gfp and yfp genes using the pCaPVX440 vector. Green fluorescence was observed in the systemic leaves from pCaPVX440-GFP and pCaPVX440-GFP-YFP infiltrated N. benthamiana at 6 dpi. (C) Detection of GFP and YFP in systemic leaves of N. benthamiana plants infiltrated with pCaPVX440-GFP (lane 2) and pCaPVX440-GFP-YFP (lane 4) at 6 dpi via Western blot analysis. Crude extract from plants infiltrated with pCaPVX440 (lanes 1 and 3) was used as a negative control. M, protein ladder. (D) Observation of GFP and YFP fluorescence in systemic leaves of plants infiltrated with pCaPVX440-GFP-YFP at 6 dpi. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.6. Western blot analysis Total soluble proteins were extracted by homogenizing the harvested leaves in an extraction buffer (50 mmol/L sodium phosphate buffer, pH 7.0, 5 mmol/L 2-mercaptoethanol, 10 mmol/L EDTA and 0.1% Triton X-100). The crude extract was centrifuged at 12,000 × g for 15 min at 4 ◦ C. Soluble proteins in the supernatant were separated in 15% SDS-PAGE gels and transferred to nitrocellulose membranes followed by probing with antibodies against GFP (Gao, 2012) or histidine-tagged-YFP (anti-His mouse monoclonal antibody) (Transgen, China), respectively. The detection was visualized using BCIP/NBT substrate as instructed by the manufacturer (Beyotime, China).
leaves by 5 days post agroinfiltration (dpi). Likewise, pCaPVX100 was able to cause systemic infection in tobacco cv. NC89, tomato cv. Micro-Tom, and potato cv. Zaodabai. The disease symptoms in these infected plants were similar to that caused by the wild type PVX, ranging from vein chlorosis to mild mosaic by 11 to 15 dpi (Fig. 1B). Virus infection in these assayed plants was further confirmed by RT-PCR at 15 dpi. Bands of 789 base pairs (bp) were amplified from the N. benthamiana, tobacco, tomato and potato plants inoculated with wild type PVX1985 and plasmid pCaPVX100, but no band was amplified from mock-inoculated plants (Fig. 1C). There were 19 nt different, resulting in 5 amino acids difference, in the genome of PVX1985 and pCaPVX100 (GenBank accession number: KJ690768) (Table S2); however, the difference had no influence on the symptoms in different host plants.
3. Results 3.1. Infectivity of pCaPVX100 in different solanaceous host plants
3.2. Expression of foreign genes in PVX-based over-expression vectors
Plasmid pCaPVX100 was introduced into leaves of N. benthamiana via agroinfiltration. The infiltrated plants started to show mosaic and epinasty symptoms in their non-infiltrated systemic
Vector pCaPVX440 (GenBank accession number: KJ690769) bearing two cloning sites was constructed to express two foreign genes simultaneously in the same cells (Fig. 2A). The gfp gene of
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Fig. 3. Schematic genome structure of pCaPVX760 and transient expression of GFP using this vector. (A) Schematic representation of pCaPVX760 and three promoters (arrows) and restriction sites in this vector. (B) Green fluorescence observed in the pCaPVX760-GFP-infiltrated N. benthamiana leaves at 2, 4, 6 and 8 dpi under UV illumination. Leaves infiltrated with pCaPVX760 were used as a negative control. (C) GFP accumulation in pCaPVX760-GFP-infiltrated N. benthamiana leaves. The leaves were collected at 2, 4, 6 and 8 dpi and detected by Western blot assay using a GFP-specific antibody. The GFP accumulation in systemic leaves of pCaPVX440-GFP-infected plants at 6 dpi was also detected. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Aequorea victoriae was cloned into pCaPVX440, in the AsiSI site, to generate pCaPVX440-GFP. By 6 dpi, strong green fluorescence was observed under UV illumination from the systemic leaves of N. benthamiana plants agroinfiltrated with pCaPVX440-GFP, but not from those infiltrated with pCaPVX440 (middle and left pictures in Fig. 2B), indicating that gfp gene was expressed correctly by the pCaPVX440-derived construct. To further confirm the functionality of this pCaPVX440 vector, we inserted the yfp gene between the SacI and MluI site and the gfp gene into AsiSI site successively to produce pCaPVX440-GFP-YFP. Introduction of pCaPVX440-GFP-YFP into N. benthamiana leaves via agroinfiltration caused mild mosaic symptom in systemic leaves by 6 dpi, similar to that caused by pCaPVX440-GFP. Under UV illumination, mixture of green and yellow fluorescence was observed from the systemic leaves of N. benthamiana plants agroinfiltrated with pCaPVX440-GFP-YFP (right picture in Fig. 2B). Both GFP and YFP fluorescence were observed in a same cell under a confocal laser scanning microscopy (Fig. 2D). In Western blotting analysis, both GFP and YFP were detected from the systemic leaves infected with pCaPVX440-GFP-YFP, using antibodies specific to GFP and histidine separately (Fig. 2C). To further increase the expression levels of foreign genes, we replaced the last 696 nt of the PVX CP ORF with a MCS (i.e. NheI, NruI and XhoI) and eliminated the three NheI sites existing in the pCaPVX440 vector through site-directed mutagenesis to produce pCaPVX760 (Fig. 3A; GenBank accession number: KJ690770). Because the PVX CP is required for cell-to-cell movement (Fedorkin et al., 2001; Lough et al., 2000), the pCaPVX760 replicon could not move systemically in plants. A gfp gene was introduced into pCaPVX760 between the NheI and NruI sites, producing pCaPVX760-GFP. The construct pCaPVX760-GFP was transformed into Agrobacterium GV3101 cells and agroinfiltrated
into N. benthamiana leaves. Green fluorescence could be observed in the infiltrated areas of N. benthamiana leaves at 2 dpi and become stronger at 4, 6 and 8 dpi (Fig. 3B). To further investigate the expression dynamics of GFP in N. benthamiana leaves, the infiltrated leaf areas were harvested at different days after infiltration, extracted, and subjected to Western blot analysis using an antibody against GFP. The GFP could be detected by 2 dpi and maintained at high expression levels at 4, 6 and 8 (Fig. 3C). The GFP expression in N. benthamiana plants infiltrated with pCaPVX760-GFP was significantly higher than that of plants infiltrated with pCaPVX440-GFP (Fig. 3C). 3.3. Silencing endogenous genes using a ligation-independent PVX-based VIGS vector To facilitate the application of PVX-based VIGS vector in highthroughput assay, we inserted ligation-independent cloning (LIC) sequence between the SacI and MluI sites in pCaPVX440 to produce pCaPVX440-LIC (Fig. 4A). Two fragments were PCR amplified from the same region of N. benthamiana pds and inserted, in the sense or antisense orientation, into pCaPVX440-LIC to produce pCaPVX440-LIC-PDS (in sense) or pCaPVX440-LIC-PDSas (‘as’ indicated antisense). Plants infiltrated with pCaPVX440-LIC-PDS developed photo-bleaching phenotype in their systemic leaves by 7 to 8 dpi (Fig. 4B). Interestingly, the silencing phenotypes caused by pCaPVX440-LIC-PDSas showed photo-bleaching phenotype one or two days earlier than that caused by the vector pCaPVX440-LICPDS. At 14 dpi, photo-bleaching was distinct on the systemic leaves of pCaPVX440-LIC-PDS and pCaPVX440-LIC-PDSas infected N. benthamiana plants, but only mosaic and epinasty symptoms were observed on systemic leaves of pCaPVX440-LIC-infected plants (Fig. 4B).
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Fig. 4. Construction of pCaPVX440-LIC vector and its application in VIGS in N. benthamiana. (A) Procedure used for construction of pCaPVX440-LIC-PDS. (B) Photo-bleaching phenotypes in leaves infected with pCaPVX440-LIC-PDS and pCaPVX440-LIC-PDSas at 14 dpi. (C) Relative accumulation levels of pds mRNA transcript in N. benthamiana plants agroinfiltrated with pCaPVX440-LIC, pCaPVX440-LIC-PDS and pCaPVX440-LIC-PDSas. RNA extracted separately from those systemic leaves was subjected to semiquantitative RT-PCR amplification (27 cycles) with the primers PDS-1-F and PDS-1-R. 1, pCaPVX440-LIC; 2, pCaPVX440-LIC-PDS; and 3, pCaPVX440-LIC-PDSas. The EF1␣ mRNA was used as an internal control (lower panel). (D) Phenotype in the systemic leaves infected with pCaPVX440-LIC-GFP at 14 dpi.
Relative accumulation of pds mRNA in those leaves was detected by semi-quantitative RT-PCR and EF1␣ mRNA was used as an internal control. EF1␣ mRNA accumulated to the same levels in plants inoculated individually with pCaPVX440-LIC, pCaPVX440LIC-PDS or pCaPVX440-LIC-PDSas. At 14 dpi, the pds mRNA level accumulated in plants inoculated with pCaPVX440-LIC-PDSas was lower than that inoculated with pCaPVX440-LIC-PDS, both of which were significantly lower than that inoculated with pCaPVX440-LIC (Fig. 4C), indicating that pCaPVX440-LIC can be used as a viral vector to silence genes in N. benthamiana plants. To further determine the silencing efficiency of this vector, we cloned the 717 bp fragment of gfp gene into pCaPVX440-LIC, producing pCaPVX440-LIC-GFP. Complete silencing of gfp gene was achieved in plants infiltrated with pCaPVX440-LIC-GFP at 14 dpi (Fig. 4D).
4. Discussion PVX is an RNA virus infecting many solanaceous plants. The region between the PVX TGB and ORF5 is flexible and has been modified for insertion of foreign sequences (Chapman et al., 1992; Lacomme and Chapman, 2008; Plchova et al., 2011). PVX-based over-expression and silencing vectors have been used successfully
to study gene functions in plants. In this paper, we report a set of PVX-based vectors that are useful in production of foreign proteins in leaves through over-expression and silencing of endogenous genes through VIGS. These vectors are introduced into plants via agroinfiltration, the cost of which is much less and the efficiency is higher than that achieved through mechanical inoculation with in vitro transcribed RNA transcripts. Many reported viral expression vectors were capable of expressing a single foreign protein in plants (Chapman et al., 1992; Shivprasad et al., 1999). Several potyvirus-based vectors were, however, modified to express two or more proteins in plants by addition of the NIa protease recognition sequence before or after the inserted foreign proteins (Dasgupta et al., 1998; Kelloniemi et al., 2008; Marcos and Beachy, 1994). A TMV-based two-component vector was also shown to express two foreign proteins in the same plant (Roy et al., 2010). BPMV-based vector, pBPMV-IA-V5, was recently modified to express a GFP and a BAR (a protein which confers the herbicide resistance to phosphinothricin) gene simultaneously in soybean (Glycine max) plants (Zhang et al., 2010). In this report we demonstrated that the pCaPVX440 vector can serve to express two different genes in N. benthamiana plants (Fig. 2B, C and D). With vector pCaPVX440 we were able to study the function of a specific gene important in plant growth or response to biotic or abiotic stresses while monitoring the gene expression
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locus using gfp or another reporter gene as a marker. This vector can also be used to study the synergistic effects of two different proteins in the same plant. It is reported that the foreign inserts in PVX-based vectors were unstable (Thomas et al., 2001). We also noticed such phenomenon with pCaPVX440 – the foreign inserts may be deleted three weeks after inoculation. However, a PVX vector carrying the coat protein gene of satellite panicum mosaic virus was stable for at least three serial systemic passages through N. benthamiana (Everett et al., 2010). The stability of pCaPVX440 will be further improved after proper modification. Gene replacement is a common strategy used in preparations of virus-based expression vectors. For examples, the CP ORF of a crucifer-infecting strain of TMV and the CP ORF of PVX were replaced with the gfp or -glucuronidase (gus) gene (Chapman et al., 1992; Marillonnet et al., 2004). Deletion of their CP ORFs resulted in mutant viruses defective in cell-to-cell movement in plant (Fedorkin et al., 2001; Lough et al., 2000). These CP deletion mutants were, however, capable of replicating in the inoculated cells after agroinfiltration (Gleba et al., 2007; Lindbo, 2007; Marillonnet et al., 2005). Similarly, the CP-less pCaPVX760 vector can serve as an expression vector and can reach a high expression level by 4 to 6 dpi (Fig. 3B). CP-less plant virus-based expression vector may have higher agroinfection rates, easier scale up, higher protein expression levels, and biocontainment/protein purification advantages (Lindbo, 2007). Moreover, the three SGPs and three MCS in pCaPVX760 vector allow the expression of two or more proteins of interest in the same cells. With these advantages, pCaPVX760 provides an alternate choice for selection of a plant viral vector to produce detectable levels of recombinant protein in plants in a very short time frame. VIGS is a useful tool for reverse genetics in plant. Silencing individual genes using virus-based vectors has facilitated identification of functions of numerous genes (Ma et al., 2012; Pang et al., 2013; Qu et al., 2012; Yuan et al., 2011). The LIC technology is known to be easy to use and thus suitable for high-throughput cloning (Dong et al., 2007; Yuan et al., 2011). Although LIC is a good alternative for current cloning strategies, only a small number of LIC-based vectors have been made available for protein production, in planta expression and silencing (De Rybel et al., 2011). To facilitate efficient gene cloning and improve the application of the PVX-based VIGS vector, we introduced a LIC sequence into the vector pCaPVX440 (Fig. 4A). As compared to the conventional cloning method, a foreign sequence insert can be easily cloned into the pCaPVX440-LIC vector at a very low cost and high efficiency. Comparing the VIGS phenotypes of pCaPVX440-LIC-GFP and pCaPVX440-LIC-PDS, the silencing efficiencies for different genes might differ significantly (Fig. 4B and D). The difference may due to the size, structure, homology (with target genes) of the inserts, and origins (endogenous genes or transgenes) of the targets (Thomas et al., 2001). With the combination of high-throughput cloning and VIGS, the Agro/LIC strategy-based pCaPVX440-LIC has potential in large-scale functional genomics of a diverse range of host plants. In summary, our new PVX-based expression vectors have the ability to over-express two different proteins in the same cells, or produce large amounts of proteins in the inoculated leaves; the LIC-based PVX VIGS vector has the advantage of high ligation efficiency and thus is competent for high throughput functional genomics analysis. These novel PVX-based expression and silencing vectors will be useful for researchers interested in rapidly expressing recombinant proteins or silencing certain genes in plants.
Acknowledgments This study was supported by Special Research Fund for Doctoral Program of Higher Education (20123702110013) from Ministry of
Education, China, National Natural Science Foundation of China (30971895 and 31011130031), Shandong Provincial Natural Science Foundation (ZR2011CM019; 110201202016; 201211-KN214). The authors are grateful to Sonya M. Baird of Mississippi State University and Xinshun Ding of Noble Foundation for critical review on the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.07.018. References Aslanidis, C., de Jong, P.J., 1990. Ligation-independent cloning of PCR products (LICPCR). Nucleic Acids Res. 18 (20), 6069–6074. Burch-Smith, T.M., Anderson, J.C., Martin, G.B., Dinesh-Kumar, S.P., 2004. Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J. 39 (5), 734–746. Burch-Smith, T.M., Schiff, M., Liu, Y., Dinesh-Kumar, S.P., 2006. Efficient virusinduced gene silencing in Arabidopsis. Plant Physiol. 142 (1), 21–27. 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Virus Research journal homepage: www.elsevier.com/locate/virusres
Development of new potato virus X-based vectors for gene over-expression and gene silencing assay Ying Wang a,b,1 , Qian-Qian Cong a,1 , Yu-Fei Lan c,1 , Chao Geng a , Xian-Dao Li a , Yuan-Cun Liang a , Zheng-You Yang b,∗∗ , Xiao-Ping Zhu a , Xiang-Dong Li a,∗ a
Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Shandong 271018, China Department of Microbiology, College of Life Sciences, Shandong Agricultural University, Shandong 271018, China c Tai’an Academy of Agricultural Sciences, Shandong 271000, China b
a r t i c l e
i n f o
Article history: Received 16 April 2014 Received in revised form 16 July 2014 Accepted 20 July 2014 Available online 27 July 2014 Keywords: Potato virus X (PVX) Over-expression Gene silencing Gene vector Ligation independent cloning
a b s t r a c t Multiple plant viruses, including potato virus X (PVX), have been modified as vectors for expressing heterologous genes or silencing endogenous genes in plants. PVX-based vectors facilitate the functional analysis of genes in plant. However, they can only express one protein in a time. In this paper we report the construction of new vectors based on a 35S promoter-driven PVX infectious clone, pCaPVX100. Vector pCaPVX440 contains two additional subgenomic promoters and can be utilized to express two foreign genes at the same time. Plasmid pCaPVX760 is a CP minus vector and can be used to express foreign proteins through the gene substitution strategy. In addition, plasmid pCaPVX100 was engineered into a gene silencing vector (pCaPVX440-LIC) by introducing a ligation independent cloning (LIC) site into the vector. These results indicate that the newly developed PVX vectors are competent for multiple research purposes. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years multiple plant RNA and DNA viruses have been modified to serve as vectors for over-expressing and/or silencing genes in plants. Some virus-based vectors were used to produce heterologous proteins or peptides with commercial importance, including antibodies or vaccine antigens, in plant cells (Gellért et al., 2012; Gleba et al., 2007; Nuzzaci et al., 2007; Roy et al., 2010). In general, plant virus-based over-expression systems are more cost-effective and easier to use than the stable transformation technology. The virus-based vectors can also be used to study gene function via virus-induced gene silencing (VIGS) (BurchSmith et al., 2004; Faivre-Rampant et al., 2004; Kumar et al., 2012; Purkayastha et al., 2010; Zhang et al., 2009, 2012). Silencing genes in plant often results in specific phenotypes that allow quick identifications of gene functions in plants. To date more than a dozen plant virus-based vectors have been used successfully to silence genes in plants, including apple latent spherical virus (Igarashi et al.,
∗ Corresponding author. Tel.: +86 (0) 538 8242523; fax: +86 (0) 538 8226399. ∗∗ Corresponding author. E-mail addresses: [email protected] (Z.-Y. Yang), [email protected], [email protected] (X.-D. Li). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.virusres.2014.07.018 0168-1702/© 2014 Elsevier B.V. All rights reserved.
2009), barley stripe mosaic virus (Yuan et al., 2011), bean pod mottle virus (BPMV) (Zhang et al., 2009, 2010), brome mosaic virus (Ding et al., 2006), cabbage leaf curl virus (Turnage et al., 2002), cotton leaf crumple virus (Tuttle et al., 2008), grapevine leafrollassociated virus-2 (Kurth et al., 2012), pea early browning virus (Constantin et al., 2004), potato virus X (PVX) (Faivre-Rampant et al., 2004; Lacomme and Chapman, 2008), tobacco mosaic virus (TMV) (Kumagai et al., 1995), tobacco rattle virus (Burch-Smith et al., 2006; Kumar et al., 2012; Ratcliff et al., 2001; Valentine et al., 2004), and tomato golden mosaic virus (Peele et al., 2001). PVX has a single-stranded genomic RNA of about 6430 nucleotides (nt) in length (Yu et al., 2010). The 5 end of the PVX RNA contains an m7 GpppG cap structure and its 3 end has a polyadenylate tail. The PVX genome has five open reading frames (ORFs). The ORF1 encodes a 164 kDa RNA-dependent RNA polymerase (RdRp) followed by three partially overlapped ORFs known as the triple gene block (TGB). The TGB encodes the 25 kDa TGBp1, the 12 kDa TGBp2, and the 8 kDa TGBp3, respectively. The last ORF in the PVX genome, ORF5, is also transcribed through a subgenomic promoter (SGP) and encodes the 25 kDa structural coat protein (CP). The TGBps and CP have been shown to function in PVX cell-to cell movement in plants (Fedorkin et al., 2001; Lough et al., 2000). PVX genome was cloned behind a T7 RNA polymerase promoter or a cauliflower mosaic virus (CaMV) 35S promoter to serve as expression and/or gene silencing vectors (Lacomme and Chapman,
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2008). The PVX-based vectors have been successfully used to transiently express foreign genes; however, they can only express one protein in a time (Cerovska et al., 2012; Chapman et al., 1992; Lacomme and Chapman, 2008; Plchova et al., 2011). PVX-based vectors have also been used to silence endogenous genes through VIGS, so as to study the function of silenced gene (Faivre-Rampant et al., 2004; Lacomme and Chapman, 2008). However, digestion and ligation procedures limit its application in large scale cloning. In this paper, we constructed new PVX-based expression vectors that can express two foreign genes simultaneously in the same cells, or express foreign proteins through the gene substitution strategy. In addition, a VIGS vector was constructed, which carries a ligation independent cloning (LIC) site and can be used in high throughout silencing studies. 2. Materials and methods 2.1. Growth of test plants Seeds of Nicotiana benthamiana, tobacco (N. tabacum) cv. NC89, tomato (Solanum lycopersicum) cv. Micro-Tom and potato (S. tuberosum) cv. Zaodabai were sown in soil. The seedlings were transplanted into pots at the 3- to 4-leaf stage, and allowed to grow in a growth chamber set at 25 ◦ C and 16 h/8 h (light/dark) conditions. 2.2. Construction of PVX infectious clone The PVX isolate 1985 (PVX1985; GenBank accession number EU571480) was from a previously published source (Yu et al., 2010) and maintained in N. tabacum plants. The PVX1985 sequence was amplified through reverse transcription (RT)-PCR from a total RNA sample extracted from infected N. tabacum leaves using a moloney murine leukemia virus (M-MuLV) reverse transcriptase (Transgen, China) and a Phusion DNA Polymerase (Thermo, Finland). To construct the full-length infectious clone of PVX1985, a 35S promoter and the 5 end 548 bp sequence of PVX genome were fused together through overlapping PCR with primer pairs 1985pro-F (‘F’ for forward) and 1985-pro-R (‘R’ for reverse), 1985-1-F and 1985-1-R (Table S1), and then ligated into vector pMD18-T simple to yield pMD18-T-1. The second fragment representing partial PVX sequence, position 543 to 3354, was amplified with the primer pair 1985-2-F and 1985-2-R. After digestion with EcoRI and XmaI enzymes, the fragment was ligated into pMD18-T-1 to yield pMD18-T-2. The 3 half PVX genome, position 3349 to the 3 -end poly (A) tail, was amplified with the primer pair 1985-3-F and 1985-3-R, digested with BamHI and XmaI, and then ligated into pMD18-T-2 to yield pMD18-T-PVX100. The full-length 35S promoter and the PVX1985 sequence were then inserted between the SalI and XmaI site in pCambia0390. The resulting vector is referred to as pCaPVX100 (Fig. 1A). The PVX100 nucleotide sequence is numbered as the PVX sequence deposited in GenBank. 2.3. Construction of PVX-based over-expression and silencing vectors To construct a pCaPVX100-based over-expression vector, we planned to introduce a multiple clone site to the region between TGB and CP ORF. However, the candidate restriction site of ApaI was also present in the plasmid pCambia0390. Therefore, we first obtained an insert containing three fragments. The first fragment contained the sequence from ApaI site (4945) to PVX CP SGP (Chapman et al., 1992; Skryabin et al., 1988) plus an AsiSI restriction site (GCG ATC GC). The second fragment contained the TMV CP SGP (Grdzelishvili et al., 2000) fused with the second cloning site (e.g. SacI, BstBI and MluI; GAG CTCGGT CCG GAG GTT CGA ACG
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ACG CGT). The third fragment contained the 3 -terminal sequence of PVX1985 genome from the PVX CP SGP to the 3 -end poly (A) tail. These three fragments were amplified separately with the primer pairs 1985-4-F/1985-7-R, 1985-6-F/1985-6-R, and 19857-F/1985-3-R, and fused by overlapping PCR with primer pair 1985-4-F and 1985-3-R. The insert was digested with ApaI and XmaI and cloned into pMD18-T-PVX100 to produce pMD18-T-PVX140. The 35S promoter sequence and the engineered PVX genome in pMD18-T-PVX140 were then cloned into pCambia0390 using the restriction sites SalI and XmaI. The thymine in the first PVX CP SGP (position 5651) and TMV CP SGP (position 5859) were changed to guanine through mutagenesis PCR to abolish the start codon. The resultant plasmid was designated as pCaPVX440 (Fig. 2A). The second over-expression vector pCaPVX760 was produced by replacing most of the PVX CP sequence (ORF5), position 6009 to 6,710, in the pCaPVX440 with three restriction sites (NheI, XhoI and NruI; GCT AGCAAA CTC GAGTTT TCG CGA). The three NheI sites in pCaPVX440 were removed by changing GCT AGC to GCA AGC (Fig. 3A). The fragments encoding the N- or C-terminal parts of an enhanced yellow fluorescent protein (eYFP) were amplified from vectors 35S-SPYNE and 35S-SPYCE (Walter et al., 2004), respectively, using the primer pairs YFP-Sac-F/Lap-YFP-R and LapYFP-F/YFP-Mlu-R (Table S1), and then used to generate the full length eyfp gene via overlapping PCR. The green fluorescent protein gene (gfp) was amplified from pTVBMV-GFP (Gao et al., 2012) through PCR using the primer pairs GFP-Asis-F/GFP-Asis-R and GFP-Nhe-F/GFP-Nru-R. The resultant gfp and eyfp fragments were inserted individually into vectors pCaPVX440 and pCaPVX760 to generate pCaPVX440-GFP, pCaPVX440-GFP-YFP and pCaPVX760GFP. Vector pCaPVX440-LIC (Fig. 4A) was produced by inserting a LIC sequence (AGG GTC TTG TCG TTC GAA CCC GAG AGG AGT A) (Aslanidis and de Jong, 1990; Dong et al., 2007; Yuan et al., 2011) containing a BstBI restriction site (underlined sequence) between the SacI and MluI sites in pCaPVX440. The pds gene was amplified with primer pairs Lic-PDS-F/Lic-PDS-R and LicPDSas-F/Lic-PDSas-R, in which the forward primers contained 5 -GGGTCTTGTCGTTCGA-3 and the reverse primers contained 5 CTCCTCTCGGGTTCGA-3 at their 5 -termini. By using primer pairs Lic-PDS-F/Lic-PDS-R and Lic-PDSas-F/Lic-PDSas-R, the same region of the pds gene was amplified, but ligated into the silencing vector pCaPVX440-LIC in different orientations. The 717 bp fragment of gfp gene was amplified with primers Lic-GFP-F and Lic-GFP-R. The PCR products were purified, and then treated at 22 ◦ C for 30 min with T4 DNA polymerase (New England Biolabs) in 1X reaction buffer containing 5 mM dTTP to generate sticky ends, and heated at 75 ◦ C for 10 min to inactivate the polymerase. The BstBI-linearized pCaPVX440-LIC vector was treated with T4 DNA polymerase in the presence of 5 mM dATP to generate sticky ends complementary to those of the PCR products mentioned above. The treated PCR products (∼200 ng) and pCaPVX440-LIC vector (∼20 ng) were mixed, incubated at 66 ◦ C for 2 min and 22 ◦ C for 10–30 min to anneal their complementary termini. Then 10 L aliquots were used for transformation into Escherichia coli DH5␣. Positive clones identified by plasmid PCR and restriction enzyme digestion were confirmed by sequencing. 2.4. Detection of the pds gene The pds gene accumulation level in N. benthamiana was detected at 14 dpi by semi-quantitative RT-PCR using primers PDS-1-F and PDS-1-R, which were complementary to regions outside the one cloned into pCaPVX440-LIC in 2.3. The semi-quantitative RT-PCR reaction was conducted in a final volume of 20 L, containing 250 ng of RNA and 0.5 M of each primer. After 27 cycles, the PCR
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Fig. 1. Analysis of pCaPVX100 infectivity in different host plants. (A) Schematic representation of pCaPVX100 and restriction sites used for cloning. 35S, the 35S promoter of CaMV; RdRp, RNA-dependent RNA polymerase; 25 kDa, 12 kDa and 8 kDa represent the proteins encoded by the triple gene block (TGB); CP, coat protein. Black arrow indicates the position of PVX CP subgenomic promoter. (B) Symptoms caused by the wild type isolate PVX1985 and the infectious clone pCaPVX100 in N. benthamiana, tobacco (N. tabacum) cv. NC89, potato (Solanum tuberosum) cv. Zaodabai and tomato (S. lycopersicum) cv. Micro-Tom at 5, 11 or 15 days post inoculation. (C) Detection of PVX CP RNA accumulation in different host plants through RT-PCR. Mo, mock inoculation; Mi, mechanical inoculation of PVX1985; Ai, agro-infiltration of pCaPVX100; M, DNA ladder.
products were analyzed in 1% agarose gels. The EF1␣ mRNA was used as an internal RNA control. 2.5. Inoculation and fluorescence visualization Plasmid pCaPVX100 and its derivatives were transformed individually into Agrobacterium tumefaciens GV3101 cells following the freeze and thaw transformation procedure (Cui et al., 1995). Overnight grown Agrobacterium cultures were pelleted through centrifugation at 5,000 × g for 3 min and then incubated in an
induction solution (10 mmol/L MES, pH 5.8, 0.1 mmol/L acetosyringone and 10 mmol/L MgCl2 ) for 3 h. Leaves of assay plants were infiltrated with individual Agrobacterium culture (OD600 = 0.5) using needle-less syringes. Leaves infiltrated with Agrobacterium harboring gfp gene-carrying constructs were examined for GFP fluorescence using a hand-held UV (365 nm wavelength) lamp (Blak Ray B100-AP lamp, UV products, Upland, CA 91786, USA). Every experiment was repeated three times independently. Three to five plants were agroinoculated by each plasmid every time.
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Fig. 2. Genomic structure of vector pCaPVX440 and its application in over-expression. (A) Schematic representation of the over-expression vector pCaPVX440. Black arrows indicate the PVX CP subgenomic promoters, green arrow indicates the position of TMV CP subgenomic promoter. (B) Over-expression of gfp and yfp genes using the pCaPVX440 vector. Green fluorescence was observed in the systemic leaves from pCaPVX440-GFP and pCaPVX440-GFP-YFP infiltrated N. benthamiana at 6 dpi. (C) Detection of GFP and YFP in systemic leaves of N. benthamiana plants infiltrated with pCaPVX440-GFP (lane 2) and pCaPVX440-GFP-YFP (lane 4) at 6 dpi via Western blot analysis. Crude extract from plants infiltrated with pCaPVX440 (lanes 1 and 3) was used as a negative control. M, protein ladder. (D) Observation of GFP and YFP fluorescence in systemic leaves of plants infiltrated with pCaPVX440-GFP-YFP at 6 dpi. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.6. Western blot analysis Total soluble proteins were extracted by homogenizing the harvested leaves in an extraction buffer (50 mmol/L sodium phosphate buffer, pH 7.0, 5 mmol/L 2-mercaptoethanol, 10 mmol/L EDTA and 0.1% Triton X-100). The crude extract was centrifuged at 12,000 × g for 15 min at 4 ◦ C. Soluble proteins in the supernatant were separated in 15% SDS-PAGE gels and transferred to nitrocellulose membranes followed by probing with antibodies against GFP (Gao, 2012) or histidine-tagged-YFP (anti-His mouse monoclonal antibody) (Transgen, China), respectively. The detection was visualized using BCIP/NBT substrate as instructed by the manufacturer (Beyotime, China).
leaves by 5 days post agroinfiltration (dpi). Likewise, pCaPVX100 was able to cause systemic infection in tobacco cv. NC89, tomato cv. Micro-Tom, and potato cv. Zaodabai. The disease symptoms in these infected plants were similar to that caused by the wild type PVX, ranging from vein chlorosis to mild mosaic by 11 to 15 dpi (Fig. 1B). Virus infection in these assayed plants was further confirmed by RT-PCR at 15 dpi. Bands of 789 base pairs (bp) were amplified from the N. benthamiana, tobacco, tomato and potato plants inoculated with wild type PVX1985 and plasmid pCaPVX100, but no band was amplified from mock-inoculated plants (Fig. 1C). There were 19 nt different, resulting in 5 amino acids difference, in the genome of PVX1985 and pCaPVX100 (GenBank accession number: KJ690768) (Table S2); however, the difference had no influence on the symptoms in different host plants.
3. Results 3.1. Infectivity of pCaPVX100 in different solanaceous host plants
3.2. Expression of foreign genes in PVX-based over-expression vectors
Plasmid pCaPVX100 was introduced into leaves of N. benthamiana via agroinfiltration. The infiltrated plants started to show mosaic and epinasty symptoms in their non-infiltrated systemic
Vector pCaPVX440 (GenBank accession number: KJ690769) bearing two cloning sites was constructed to express two foreign genes simultaneously in the same cells (Fig. 2A). The gfp gene of
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Fig. 3. Schematic genome structure of pCaPVX760 and transient expression of GFP using this vector. (A) Schematic representation of pCaPVX760 and three promoters (arrows) and restriction sites in this vector. (B) Green fluorescence observed in the pCaPVX760-GFP-infiltrated N. benthamiana leaves at 2, 4, 6 and 8 dpi under UV illumination. Leaves infiltrated with pCaPVX760 were used as a negative control. (C) GFP accumulation in pCaPVX760-GFP-infiltrated N. benthamiana leaves. The leaves were collected at 2, 4, 6 and 8 dpi and detected by Western blot assay using a GFP-specific antibody. The GFP accumulation in systemic leaves of pCaPVX440-GFP-infected plants at 6 dpi was also detected. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Aequorea victoriae was cloned into pCaPVX440, in the AsiSI site, to generate pCaPVX440-GFP. By 6 dpi, strong green fluorescence was observed under UV illumination from the systemic leaves of N. benthamiana plants agroinfiltrated with pCaPVX440-GFP, but not from those infiltrated with pCaPVX440 (middle and left pictures in Fig. 2B), indicating that gfp gene was expressed correctly by the pCaPVX440-derived construct. To further confirm the functionality of this pCaPVX440 vector, we inserted the yfp gene between the SacI and MluI site and the gfp gene into AsiSI site successively to produce pCaPVX440-GFP-YFP. Introduction of pCaPVX440-GFP-YFP into N. benthamiana leaves via agroinfiltration caused mild mosaic symptom in systemic leaves by 6 dpi, similar to that caused by pCaPVX440-GFP. Under UV illumination, mixture of green and yellow fluorescence was observed from the systemic leaves of N. benthamiana plants agroinfiltrated with pCaPVX440-GFP-YFP (right picture in Fig. 2B). Both GFP and YFP fluorescence were observed in a same cell under a confocal laser scanning microscopy (Fig. 2D). In Western blotting analysis, both GFP and YFP were detected from the systemic leaves infected with pCaPVX440-GFP-YFP, using antibodies specific to GFP and histidine separately (Fig. 2C). To further increase the expression levels of foreign genes, we replaced the last 696 nt of the PVX CP ORF with a MCS (i.e. NheI, NruI and XhoI) and eliminated the three NheI sites existing in the pCaPVX440 vector through site-directed mutagenesis to produce pCaPVX760 (Fig. 3A; GenBank accession number: KJ690770). Because the PVX CP is required for cell-to-cell movement (Fedorkin et al., 2001; Lough et al., 2000), the pCaPVX760 replicon could not move systemically in plants. A gfp gene was introduced into pCaPVX760 between the NheI and NruI sites, producing pCaPVX760-GFP. The construct pCaPVX760-GFP was transformed into Agrobacterium GV3101 cells and agroinfiltrated
into N. benthamiana leaves. Green fluorescence could be observed in the infiltrated areas of N. benthamiana leaves at 2 dpi and become stronger at 4, 6 and 8 dpi (Fig. 3B). To further investigate the expression dynamics of GFP in N. benthamiana leaves, the infiltrated leaf areas were harvested at different days after infiltration, extracted, and subjected to Western blot analysis using an antibody against GFP. The GFP could be detected by 2 dpi and maintained at high expression levels at 4, 6 and 8 (Fig. 3C). The GFP expression in N. benthamiana plants infiltrated with pCaPVX760-GFP was significantly higher than that of plants infiltrated with pCaPVX440-GFP (Fig. 3C). 3.3. Silencing endogenous genes using a ligation-independent PVX-based VIGS vector To facilitate the application of PVX-based VIGS vector in highthroughput assay, we inserted ligation-independent cloning (LIC) sequence between the SacI and MluI sites in pCaPVX440 to produce pCaPVX440-LIC (Fig. 4A). Two fragments were PCR amplified from the same region of N. benthamiana pds and inserted, in the sense or antisense orientation, into pCaPVX440-LIC to produce pCaPVX440-LIC-PDS (in sense) or pCaPVX440-LIC-PDSas (‘as’ indicated antisense). Plants infiltrated with pCaPVX440-LIC-PDS developed photo-bleaching phenotype in their systemic leaves by 7 to 8 dpi (Fig. 4B). Interestingly, the silencing phenotypes caused by pCaPVX440-LIC-PDSas showed photo-bleaching phenotype one or two days earlier than that caused by the vector pCaPVX440-LICPDS. At 14 dpi, photo-bleaching was distinct on the systemic leaves of pCaPVX440-LIC-PDS and pCaPVX440-LIC-PDSas infected N. benthamiana plants, but only mosaic and epinasty symptoms were observed on systemic leaves of pCaPVX440-LIC-infected plants (Fig. 4B).
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Fig. 4. Construction of pCaPVX440-LIC vector and its application in VIGS in N. benthamiana. (A) Procedure used for construction of pCaPVX440-LIC-PDS. (B) Photo-bleaching phenotypes in leaves infected with pCaPVX440-LIC-PDS and pCaPVX440-LIC-PDSas at 14 dpi. (C) Relative accumulation levels of pds mRNA transcript in N. benthamiana plants agroinfiltrated with pCaPVX440-LIC, pCaPVX440-LIC-PDS and pCaPVX440-LIC-PDSas. RNA extracted separately from those systemic leaves was subjected to semiquantitative RT-PCR amplification (27 cycles) with the primers PDS-1-F and PDS-1-R. 1, pCaPVX440-LIC; 2, pCaPVX440-LIC-PDS; and 3, pCaPVX440-LIC-PDSas. The EF1␣ mRNA was used as an internal control (lower panel). (D) Phenotype in the systemic leaves infected with pCaPVX440-LIC-GFP at 14 dpi.
Relative accumulation of pds mRNA in those leaves was detected by semi-quantitative RT-PCR and EF1␣ mRNA was used as an internal control. EF1␣ mRNA accumulated to the same levels in plants inoculated individually with pCaPVX440-LIC, pCaPVX440LIC-PDS or pCaPVX440-LIC-PDSas. At 14 dpi, the pds mRNA level accumulated in plants inoculated with pCaPVX440-LIC-PDSas was lower than that inoculated with pCaPVX440-LIC-PDS, both of which were significantly lower than that inoculated with pCaPVX440-LIC (Fig. 4C), indicating that pCaPVX440-LIC can be used as a viral vector to silence genes in N. benthamiana plants. To further determine the silencing efficiency of this vector, we cloned the 717 bp fragment of gfp gene into pCaPVX440-LIC, producing pCaPVX440-LIC-GFP. Complete silencing of gfp gene was achieved in plants infiltrated with pCaPVX440-LIC-GFP at 14 dpi (Fig. 4D).
4. Discussion PVX is an RNA virus infecting many solanaceous plants. The region between the PVX TGB and ORF5 is flexible and has been modified for insertion of foreign sequences (Chapman et al., 1992; Lacomme and Chapman, 2008; Plchova et al., 2011). PVX-based over-expression and silencing vectors have been used successfully
to study gene functions in plants. In this paper, we report a set of PVX-based vectors that are useful in production of foreign proteins in leaves through over-expression and silencing of endogenous genes through VIGS. These vectors are introduced into plants via agroinfiltration, the cost of which is much less and the efficiency is higher than that achieved through mechanical inoculation with in vitro transcribed RNA transcripts. Many reported viral expression vectors were capable of expressing a single foreign protein in plants (Chapman et al., 1992; Shivprasad et al., 1999). Several potyvirus-based vectors were, however, modified to express two or more proteins in plants by addition of the NIa protease recognition sequence before or after the inserted foreign proteins (Dasgupta et al., 1998; Kelloniemi et al., 2008; Marcos and Beachy, 1994). A TMV-based two-component vector was also shown to express two foreign proteins in the same plant (Roy et al., 2010). BPMV-based vector, pBPMV-IA-V5, was recently modified to express a GFP and a BAR (a protein which confers the herbicide resistance to phosphinothricin) gene simultaneously in soybean (Glycine max) plants (Zhang et al., 2010). In this report we demonstrated that the pCaPVX440 vector can serve to express two different genes in N. benthamiana plants (Fig. 2B, C and D). With vector pCaPVX440 we were able to study the function of a specific gene important in plant growth or response to biotic or abiotic stresses while monitoring the gene expression
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locus using gfp or another reporter gene as a marker. This vector can also be used to study the synergistic effects of two different proteins in the same plant. It is reported that the foreign inserts in PVX-based vectors were unstable (Thomas et al., 2001). We also noticed such phenomenon with pCaPVX440 – the foreign inserts may be deleted three weeks after inoculation. However, a PVX vector carrying the coat protein gene of satellite panicum mosaic virus was stable for at least three serial systemic passages through N. benthamiana (Everett et al., 2010). The stability of pCaPVX440 will be further improved after proper modification. Gene replacement is a common strategy used in preparations of virus-based expression vectors. For examples, the CP ORF of a crucifer-infecting strain of TMV and the CP ORF of PVX were replaced with the gfp or -glucuronidase (gus) gene (Chapman et al., 1992; Marillonnet et al., 2004). Deletion of their CP ORFs resulted in mutant viruses defective in cell-to-cell movement in plant (Fedorkin et al., 2001; Lough et al., 2000). These CP deletion mutants were, however, capable of replicating in the inoculated cells after agroinfiltration (Gleba et al., 2007; Lindbo, 2007; Marillonnet et al., 2005). Similarly, the CP-less pCaPVX760 vector can serve as an expression vector and can reach a high expression level by 4 to 6 dpi (Fig. 3B). CP-less plant virus-based expression vector may have higher agroinfection rates, easier scale up, higher protein expression levels, and biocontainment/protein purification advantages (Lindbo, 2007). Moreover, the three SGPs and three MCS in pCaPVX760 vector allow the expression of two or more proteins of interest in the same cells. With these advantages, pCaPVX760 provides an alternate choice for selection of a plant viral vector to produce detectable levels of recombinant protein in plants in a very short time frame. VIGS is a useful tool for reverse genetics in plant. Silencing individual genes using virus-based vectors has facilitated identification of functions of numerous genes (Ma et al., 2012; Pang et al., 2013; Qu et al., 2012; Yuan et al., 2011). The LIC technology is known to be easy to use and thus suitable for high-throughput cloning (Dong et al., 2007; Yuan et al., 2011). Although LIC is a good alternative for current cloning strategies, only a small number of LIC-based vectors have been made available for protein production, in planta expression and silencing (De Rybel et al., 2011). To facilitate efficient gene cloning and improve the application of the PVX-based VIGS vector, we introduced a LIC sequence into the vector pCaPVX440 (Fig. 4A). As compared to the conventional cloning method, a foreign sequence insert can be easily cloned into the pCaPVX440-LIC vector at a very low cost and high efficiency. Comparing the VIGS phenotypes of pCaPVX440-LIC-GFP and pCaPVX440-LIC-PDS, the silencing efficiencies for different genes might differ significantly (Fig. 4B and D). The difference may due to the size, structure, homology (with target genes) of the inserts, and origins (endogenous genes or transgenes) of the targets (Thomas et al., 2001). With the combination of high-throughput cloning and VIGS, the Agro/LIC strategy-based pCaPVX440-LIC has potential in large-scale functional genomics of a diverse range of host plants. In summary, our new PVX-based expression vectors have the ability to over-express two different proteins in the same cells, or produce large amounts of proteins in the inoculated leaves; the LIC-based PVX VIGS vector has the advantage of high ligation efficiency and thus is competent for high throughput functional genomics analysis. These novel PVX-based expression and silencing vectors will be useful for researchers interested in rapidly expressing recombinant proteins or silencing certain genes in plants.
Acknowledgments This study was supported by Special Research Fund for Doctoral Program of Higher Education (20123702110013) from Ministry of
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