SBWMV 19K protein is a conserved CRP

The Pfam Protein Families Database reports a family of CRPs with similar sequences which includes proteins from BSMV, PSLV, PCV and SBWMV (Pfam 04521.5). Since there are viruses not included in the Pfam report that encode CRPs, this study was undertaken to determine if there is a larger CRP family containing related viral proteins. Further examination in this study reveals that the CRPs encoded by all known hordei-, peclu- and furoviruses share significant sequence similarity (Fig. 1). Efforts to find similarity between these proteins and CRPs encoded by pomo-, beny- and potyviruses were not successful. Whether these other plant viral CRPs are also suppressors of silencing can not be concluded at this point for two reasons: insufficient study and only weak sequence similarity relationships. Sequences of CRPs that affect virus replication and are encoded by members of other virus genera were also determined to be unrelated [25].

The SBWMV 19K protein is a CRP because it contains nine Cys residues [20]. Seven of these Cys residues are conserved in all furovirus proteins and are located in the N-terminal half of the protein. Five of these residues are within the block of sequences designated as protein family Pfam04521.5 and three of the conserved Cys residues are also conserved in the hordeiviral and pecluviral proteins. A selection from this alignment was corrected for several misplacements of short peptide sequences and is shown in Figure 1. The alignment represents the entire length of these proteins, although the termini are aligned with less confidence than the core. Examination of the tobraviral CRP sequences revealed sufficient similarity to justify their alignment with the Pfam04521.5 sequences. The alignment resulted in a significance score between 6 and 7, suggesting that the tobraviral proteins belong to this family.

The multiple sequence alignment of 33 CRPs from furo-, tobra-, peclu-, and hordeiviruses (Fig. 1) revealed three absolutely conserved residues: Cys70, Cys112, and His116 (numbering based on the aligned sequences). Gly113 was conserved in all viruses (except TRV-CAN) and is contained within a Cys-Gly-Xaa-Xaa-His motif in which one of the two Xaa residues is Lys or Arg. There is a Cys residue at position 7, 8 or 9 which is conserved in all except PCV and IPCV (pecluvirus) amino acid sequences. Alignment of the N-terminus is not exact since the PCV and IPCV proteins are N-terminally truncated. Within the N-terminal half, there are additional positions containing Cys residues that are conserved for some but not all viruses. For example, Cys9 is conserved among hordei-, tobra-, and some furoviruses; Cys at position 32 and 33 is conserved among all but pecluviruses; Cys36 is conserved among hordei- and furoviruses; Cys45 is conserved among furo and tobraviruses; Cys76 is conserved among furo and tobraviruses (except for SCSV; the pecluvirus PCV, but not IPCV, also has Cys76); Cys80 is conserved among all viruses except PeRSV and PEBV. Lys at position 52 and Arg at position 54 or 55 (Lys-Xaa-Arg or Lys-Xaa-Xaa-Arg) are conserved among all except PSLV. Gly at position 77 is conserved among all except tobraviruses.

The secondary structure prediction derived from the multiple sequence alignment is a long helical region extending from or slightly beyond the Cys-Gly-Xaa-Xaa-His motif to within 20 residues of the C-terminus. The furoviral proteins have spacings of conserved Leu residues from positions 89 to 106 consistent with a leucine zipper structure (which was not apparent in the original Pfam 04521.5). The N-terminal halves of the aligned amino acid sequences, containing most of the Cys residues, have a mixture of extended, helical and loop predicted structures.

The pecluviruses PCV and IPCV, and the hordeiviruses BSMV, LyRSV, and PSLV proteins contain a Ser-Lys-Leu sequence at the C-terminus. This tripeptide was shown for PCV to be a peroxisomal targeting signal [16]. This signal is not present in CRPs of furo- or tobraviruses.

SBWMV 19K CRP aggravates PVX-induced symptoms

The tobravirus and hordeivirus CRPs have been described as pathogenicity determinants that regulate symptom severity in infected plants [15]. Since the SBWMV 19K protein is a similar CRP, experiments were conducted to determine if it also has an effect on symptom expression. The SBWMV 19K ORF was inserted into the PVX genome and PVX.19K infectious transcripts were used to inoculate N. benthamiana, N. clevelandii, C. quinoa, and C. amaranticolor leaves (Fig. 2). As a control, plants were also inoculated with PVX.GFP, which has the green fluorescent protein (GFP) gene inserted into the viral genome. The spread of PVX.GFP expression was monitored using a handheld UV lamp to monitor GFP expression and verify systemic virus accumulation (data not shown).

Symptoms were first observed in plants inoculated with PVX.GFP and PVX.19K between 10 and 14 dpi. By 21 dpi, systemic necrosis was evident in N. benthamiana and N. clevelandii plants inoculated with PVX.19K (Fig. 2A, B and 2D) while PVX.GFP infected plants showed systemic mosaic symptoms (Fig. 2A and 2C). N. benthamiana plants infected with PVX.19K were clearly stunted in comparison to plants infected with PVX.GFP (Fig. 2A). The PVX.19K infected N. clevelandii leaves collapsed by 21 dpi (Fig. 2D).

Immunoblot and northern analyses were conducted to verify PVX accumulation in the upper leaves of N. benthamiana plants. Immunoblot analysis was conducted using anti-PVX CP serum. High levels of PVX CP was detected in plants that were systemically infected with PVX.GFP (Fig. 3A lanes 1–4) and PVX.19K (Fig. 3A lanes 5–8). The SBWMV 19K CRP had no obvious effect on PVX accumulation in upper noninoculated leaves. Viral RNA accumulation was analyzed by northern blot and high levels of genomic RNA was detected in the upper leaves of PVX.GFP (Figure 3B lanes 2–4) and PVX.19K (Fig. 3B lanes 5–8) inoculated plants. Thus, the SBWMV 19K CRP did not seem to have a deleterious effect on PVX accumulation. RT-PCR was used to verify that the SBWMV 19K ORF was maintained in the PVX genome in systemically infected plants. RNA samples taken from the upper leaves of N. benthamiana plants which were used for northern analysis, were also used in RT-PCR reactions to verify the presence of the SBWMV 19K ORF in the PVX genome. In all samples it appeared that the SBWMV 19K CRP was stably maintained in the PVX genome (data not shown).

PVX.19K produced large necrotic lesions in the C. quinoa and C. amaranticolor leaves. Local lesions were detected in plants inoculated with PVX.GFP or PVX.19K between 5 and 7 dpi. PVX.19K-inoculated C. quinoa plants showed severe necrotic local lesions (Fig. 2E). The necrotic lesions gradually merged and the infected tissue eventually collapsed. PVX.19K-inoculated C. amaranticolor leaves showed enlarged chlorotic lesions advancing to necrotic lesions over time (Fig. 2F). PVX.GFP-inoculated C. quinoa leaves showed small chlorotic and necrotic local lesions while PVX.GFP-inoculated C. amaranticolor leaves showed mild flecks (Fig. 2F). Association of PVX.GFP with the local lesions was verified using a hand held UV lamp (data not shown).

SBWMV 19K CRP is a suppressor of RNA silencing

In this study we employed a widely used "reversal of silencing assay" to determine if the SBWMV 19K CRP is a suppressor of RNA silencing in plants [28]. In this assay, GFP-expression in the 16C transgenic N. benthamiana plants (Fig. 4B) was silenced by infiltrating young leaves with a suspension of Agrobacterium expressing GFP. The progression of GFP silencing was viewed first locally and then systemically using a hand held UV lamp. Within two weeks, the spread of GFP silencing was viewed systemically (Fig. 4C) and by three weeks, the only visible fluorescence is red fluorescence due to chlorophyll (Fig. 4D). At this time, the silenced plants were inoculated with PVX.19K. As PVX.19K viruses spread locally and then systemically, there was no change in GFP expression in the inoculated leaves or in the upper leaves (Fig. 4E). However, GFP expression was observed in the emerging leaves (Fig. 4F – H). The SBWMV 19K CRP prevented RNA silencing only in emerging leaves where RNA silencing had not developed prior to virus infection. As a control, plants were also inoculated with PVX.GUS following infiltration with Agrobacterium. There was no evidence of GFP expression in the inoculated, mature, or new emerging leaves. The silencing phenotype was unaffected by PVX.GUS.

Northern analyses was conducted to confirm RNA silencing in the upper leaves of Agrobacterium-infiltrated leaves and in the plants inoculated with PVX.GUS (Fig. 4I and 4J). GFP specific RNAs were detected in transgenic plants (Figure 4I lanes 4–7) and emerging leaves of plants injected with Agrobacterium and inoculated with PVX.19K (Figure 4J lanes 1–4). GFP specific RNAs were not detected in untreated nontransgenic plants (Figure 4I lanes 1–3) or in plants that were injected with Agrobacterium and inoculated with PVX.GUS (lanes 4–8). RNA samples collected from non silenced and silenced plants were also tested by Northern analysis to confirm the systemic accumulation of PVX.GUS or PVX.19K (data not shown). Since, GFP expression was restored in plants systemically infected with PVX.19K but remained silenced in plants inoculated with PVX.GUS, it is likely that the SBWMV 19K ORF is a suppressor of RNA silencing.

Amino acid sequence comparisons

Related protein sequences were identified and retrieved from the NCBI data bank using PSI-BLAST. A PSI-BLAST search was launched with the amino acid sequence of the 19K CRP of Chinese wheat mosaic virus (CWMV, a furovirus). A similar search began with the amino acid sequence of BSMV gamma b, a sequence recovered in the CWMV search. Both searches converged at the second iteration and retrieved the same set of 22 sequences. This set contained CRPs derived from furo-, peclu- and hordeiviruses and contained the conserved P18 PFAM domain ("protein family"URL reference http://pfam.wustl.edu/[36].

A preliminary alignment of the retrieved proteins sequences was performed using the multiple sequence alignment mode of ClustalX. These twenty two furovirus and hordeivirus sequences were aligned using ClustalX alignments suggested in the BLAST outputs and PFAM. The tobraviral CRPs were not recovered by the above procedure, but upon manual inspection, appeared to have Cys residues in a linear arrangement that was similar to the set of 22 proteins. Eleven tobraviral protein sequences, exclusive members of a conserved domain in the Conserved Domain database http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml were aligned using ClustalX [37]. This tobraviral amino acid sequence alignment and the alignment of the 22 amino acid sequences sequences were assembled by ClustalX in profile mode, followed by manual adjustment. Amino acid sequences of aligned furo- and hordeiviral proteins were aligned with tobraviral amino acid sequences in profile mode of ClustalX (a total of thirty three sequences were aligned). A total of 33 amino acid sequences were aligned. In all cases, adjustments to the alignments were made using Se-Al [38]. Significance scores for the alignment of the two groups of protein sequences were calculated as previously described, using a structural conservation matrix, SCM2, for scoring [39].

Plasmids and bacterial strain

All plasmids were used to transform Escherichia coli strain JM109 [40]. The plasmids pPVX.GFP is an infectious viral clone and contains a bacteriophage T7 promoter [39]. The pPVX.GFP plasmid contains the PVX genome and the GFP adjacent to a duplicated CP subgenomic promoter. The plasmid pHST2-14 contains the TBSV genome and a mutation in the TBSV P19 ORF eliminating expression of a protein that suppresses RNA silencing [10, 42]. The plasmid pTBSV.GFP contains GFP inserted into the TBSV genome replacing the viral CP ORF [10].

The SBWMV 19K CRP ORF was inserted into the PVX.GFP genome, replacing the GFP ORF. The 19K CRP ORF was reverse transcribed and PCR amplified from purified SBWMV RNA using a forward primer (GCG GGG ATC GAT ATG TCT ACT GTT GGT TTC CAC) containing added sequences encoding a Cla I restriction site (underlined) and a reverse primer (CGC GTC GAC TCA CAA AGA GGA TAT CTT CTT TGG C) containing sequences encoding a Sal I restriction site (underlined). PCR products and pPVX.GFP plasmids were digested with Cla I and Sal I and then were ligated to prepare pPVX.19K.

In vitro transcription and plant inoculations

In vitro transcription reactions contained: 0.25 μg of linearized DNA, 5 μl of 5X T7 transcription buffer, 1.0 μl of 0.1 M DTT, 0.5 μl of SUPERase·In™ ribonuclease inhibitor (20 U/ μl) (Ambion, Austin, TX), 2.5 μl of an NTP mixture containing 5 mM ATP, CTP, UTP, and GTP (Pharmacia-Pfizer, Mississauga, Ontario, Canada), 0.7 μl of T7 polymerase (Ambion), and nuclease-free water to a final volume of 25 μl. The reactions were incubated for one and a half hour at 37°C [10].

Nicotiana benthamiana, N. clevelandii, Chenopodium quinoa, and C. amaranticolor plants were inoculated with infectious transcripts to study disease severity. Four plants, two leaves per plant, were inoculated in each experiment. Experiments were repeated at least three times. Ten μl of undiluted PVX.GFP or PVX.19K transcripts were rub-inoculated to each plant.

The transgenic N. benthamiana line 16C was used to study RNA silencing. This line is homozygous for the GFP transgene at a single locus [44]. Plants were inoculated with transcripts following infiltration with Agrobacterium (see below).

Agrobacterium infiltration of leaves

Agrobacterium tumefaciens strain C58C1 (pCH32) carrying a binary plasmid expressing GFP from a Cauliflower mosaic virus (CaMV) 35S promoter was used to silence GFP expression in N. benthamiana line 16C. Agrobacterium cultures were grown overnight at 28°C in 5 ml of L-broth medium containing 5 μg/ml of tetracycline and 50 μg/ml of kanamycin. This 5 ml culture was used to inoculate 50 ml L-broth and grown overnight in medium containing 5 μg/ml tetracycline, 50 μg/ml kanamycin, 10 mM MES, and 20 μM acetosyringone. Cultures of Agrobacterium containing GFP were pelleted by centrifugation and resuspended in a solution containing 10 mM MgCl₂, 10 mM MES, and 150 μM acetosyringone. The final concentration of Agrobacterium was 0.5 OD₆₀₀. The suspension was left at room temperature for 2–3 hours and then loaded into a 2 ml syringe. The syringe was used to infiltrate the suspension into the underside of the leaf.

Visualization of GFP

A hand-held model B-100 BLAK-RAY long wave ultraviolet lamp (Ultraviolet Products, Upland, CA) was used to monitor GFP expression in 16C plants infiltrated with Agrobacterium and in PVX.GFP inoculated plants. GFP fluorescence was recorded with a Sony Digital Still Camera model DSC-F717 (Sony Corporation of America, New York City, New York). In all plants analyzed, GFP expression was monitored every 3 days for up to 21 days post inoculation (dpi) or post infiltration with Agrobacterium.

Immunoblot analysis

Immunoblot analyses were conducted according to [40]. Total protein from uninfected and infected N. benthamiana leaves was extracted in 1:10 (w/v) grinding buffer (100 mM Tris-HCl pH 7.50, 10 mM KCl, 5 mM MgCl₂, 400 mM sucrose, 10% glycerol, and 10 mM β-mercaptoethanol). Extracts were centrifuged at 10,000 g for 10 min. Equal volumes of supernatants and protein loading buffer (2 % SDS, 0.1 M dithiothreitol, 50 mM Tris-HCl pH 6.8, 0.1% bromophenol blue, and 10 % glycerol) were mixed and boiled for 5 min. SDS-PAGE was carried out for 1 h at 200 V using 30 μl of each sample and 12.5% SDS -PAGE and the Biorad Mini-Protean 3 system (Biorad Laboratories, Hercules, CA). Proteins were transferred to PVDF membranes (Amersham Biosciences Corp., Piscataway, NJ) at 4°C overnight using protein transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, and 20% methanol, pH 8.3) and a BioRad Trans-Blot system (BioRad Laboratories). Immunoblot analyses were conducted using the ECL-Plus Western Blotting Detection Kit (Amersham Biosciences Corp.). PVX CP antiserum (1:200) (Agdia, Elkhart, IN) was used.

Northern analysis

Northern analyses were conducted according to [40]. For analyses of PVX infected plants and GFP expressing transgenic plants, a radiolabeled DNA probe was prepared using Rediprime II Random Prime Labeling System (Amersham Biosciences Corp.). Labeling was conducted using PCR products corresponding to either the GFP or PVX CP ORFs.

For detection of TBSV.GFP and TBSV.19K in infected plant extracts, a DNA probe was labeled with digoxigenin (DIG). TBSV.GFP plasmids were digested with Nco I and Sal I and a 614 nt fragment was gel eluted and labeled using Dig High Prime kit (Roche Applied Science Inc. Indianapolis, IN). The CSPD DIG Luminescence Detection Kit (Roche Applied Science Inc.) was used for chemiluminescence detection of DIG-labeled probes. Special thanks to Wenping Qui at Southwest Missouri State University for assistance with studies using TBSV to express the SBWMV 19k.

The p26SBE-2 plasmid was obtained from Kay Scheets at Oklahoma State University and contains the 26S ribosomal RNA gene in pBluescript. This plasmid was used to prepare a DNA probe for membrane detection of rRNA [45]. The p26SBE-2 plasmid was digested with Bam HI and Eco RI and a 1 kb fragment corresponding to the 26S rRNA was recovered and labeled using the Dig High Prime DNA labeling system (Roche Applied Science Inc.).