Cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant

Abstract

Little is known about how plant viruses of a single species exhibit different movement behavior in different host species. Two Cymbidium mosaic potexvirus (CymMV) isolates, M1 and M2, were studied. Both can infect Phalaenopsis orchids, but only M1 can systemically infect Nicotiana benthamiana plants. Protoplast inoculation and whole-mount in situ hybridization revealed that both isolates can replicate in N. benthamiana; however, M2 was restricted to the initially infected cells. Genome shuffling between M1 and M2 revealed that two control modes are involved in CymMV host dependent movement. The M1 coat protein (CP) plays a dominant role in controlling CymMV movement between cells, because all chimeric CymMV viruses containing the M1 CP systemically infected N. benthamiana plants. Without the M1 CP, one chimeric virus containing the combination of the M1 triple gene block proteins (TGBps), the M2 5' RNA (1-4333), and the M2 CP effectively moved in N. benthamiana plants. Further complementation analysis revealed that M1 TGBp1 and TGBp3 are co-required to complement the movement of the chimeric viruses in N. benthamiana. The amino acids within the CP, TGBp1 and TGBp3 which are required or important for CymMV M2 movement in N. benthamiana plants were mapped. The required amino acids within the CP map to the predicted RNA binding domain. RNA-protein binding assays revealed that M1 CP has higher RNA binding affinity than does M2 CP. Yeast two-hybrid assays to detect all possible interactions of M1 TGBps and CP, and only TGBp1 and CP self-interactions were observed.

Fig. 1

Schematic representation of CymMV cDNA infectious clones (A) and detection of CymMV in infected Phalaenopsis amabilis (B) and Nicotiana benthamiana (C) by RT-PCR. (A) Rectangles represent open reading frames encoded by CymMV genomic RNA. RNA-dependent RNA polymerase (RdRp), triple gene block ORFs 1, 2, and 3, capsid protein (CP) and green fluorescent protein (GFP). The mutated sequences in pCymMV-R- are indicated by bold letters. Scale bar, in nucleotides, is shown at the bottom. P. amabilis var. formosa (B) and N. benthamiana (C) inoculated with buffer (H), pCymMV-R- transcripts (R-), pCymMV-M1 transcripts (M1) and pCymMV-M2 transcripts (M2), with CymMV-infected Phalaenopsis used as a positive control (D). Total nucleic acids were extracted from CymMV-inoculated and upper leaves distal from the inoculation point, and CymMV was detected by RT-PCR. pCymMV-R- is a replication-incompetent clone used as a negative control (see Materials and Methods). Numbers at the left correspond to positions of marker DNAs (M) (sizes in 1000 base pairs).

Fig. 2

Protoplast inoculation assay and whole-mount in situ hybridization. (A) A total of 5×10⁵N. benthamiana protoplasts were inoculated with water (Mock) and transcripts of pCymMV-M1-GFP and pCymMV-M2-GFP. Cells were examined 14 h post-inoculation by light and fluorescent microscopy. (B) Leaves were first fixed with 1× phosphate buffered saline solution (pH 7.2) containing 0.1% Tween 20, 0.08 M EGTA, 10% DMSO and 5% paraformaldehyde then hybridized with a Dig-labeled CymMV CP probe. The mock, CymMV-M1 and CymMV-M2 infected leaves are indicated. The white rectangle indicates the region selected for further zooming. Magnification and scale bars in micrometers (μm) are indicated. The red “jigsaw shaped” pattern indicates a single epidermal cell.

Fig. 3

Schematic representation of genome organization and infectivity assay of the parental CymMV-M1 and CymMV-M2 and the derived chimeric constructs. (A–H). Rectangles represent open reading frames encoded by CymMV genomic RNA, RNA-dependent RNA polymerase (RdRp), triple gene block (TGB) ORFs 1, 2, and 3 and capsid protein (CP). Sequences corresponding to pCymMV-M1 and pCymMV-M2 are indicated by gray and white rectangles, respectively. The restriction enzyme sites for constructing chimeric viruses are indicated. (Although HpaI sites are located 129 nt downstream of CP translation start sites, the amino acid sequences in the regions between M1 and M2 are identical.) Clones competent in protoplast accumulation and systemic infection in N. benthamiana are indicated by +, and the ratio of systemic infected to total inoculated plants is indicated. Systemic infection was detected 2 weeks post-inoculation by RT-PCR. (I) Protoplast infectivity was detected 24 h post-inoculation by northern blot hybridization, and the ribosomal RNA used for a loading control are indicated. Genomic RNA (G), TGBp, and CP subgenomic RNA are indicated. The pCymMV-R- used as a negative control is illustrated in Fig. 1. The average percentage of relative real-time RT-PCR quantification (from 3 independent experiments) of CymMV RNA from CymMV clone-infected protoplasts at 24 h post-inoculation is indicated. The accumulation of pCymMV-M1 was set at 100% for relative quantification. Numbers at the left correspond to positions of marker RNAs (sizes in 1000 nucleotides) analyzed in the same gel.

Fig. 4

Schematic representation of genome organization and infectivity assay of the parental CymMV-M1 and CymMV-M2 and the derived TGBp1 and TGBp3 chimeric constructs in N. benthamiana. (A–H). Sequences corresponding to pCymMV-M1 and pCymMV-M2 are indicated by gray and white rectangles, respectively. Clones competent in protoplast accumulation and systemic infection are indicated by +, and the ratio of systemic infected to total inoculated plants is indicated. Systemic infection was detected 2 weeks post-inoculation by RT-PCR. (I) Protoplast infectivity was detected 24 h post-inoculation by northern blot hybridization, and the ribosomal RNA used for a loading control are indicated. Genomic RNA (G), TGBp, and CP subgenomic RNA are indicated. The pCymMV-R- used as a negative control is illustrated in Fig. 1. The average percentage of relative real-time RT-PCR quantification (from 3 independent experiments) of CymMV RNA from CymMV clone-infected protoplasts at 24 h post-inoculation is indicated below the gels. The accumulation of pCymMV-M1 was set at 100% for relative quantification. Numbers at the left correspond to positions of marker RNAs (sizes in 1000 nucleotides) analyzed in the same gel. The primers CymMV F3783 and CymMV CPR used in construction all chimeric viruses are indicated. Other primers used in construction individual chimeric viruses are in Supplementary Table S1. Numbers at the left correspond to positions of marker RNAs (sizes in 1000 nucleotides) analyzed in the same gel.

Fig. 5

Model of dominant and matching control modes in Cymbidium mosaic potexvirus movement between cells. The M1 CP plays a dominant role in controlling the trafficking of the infection between cells. However, besides the CP-mediated dominant control, another control mode that requires correct matching of TGBp1, TGBp3, CP and 5' RNA also allowed the movement of CymMV between cells. The red and blue lines indicate the functional combination of movement accessory components in dominant and matching control modes, respectively. The gray lines indicated the incorrect matching of movement accessory components. The M1 and M2 RNAs, TGBp1s, TGBp2s, TGBp3s and RNA-dependent RNA polymerase (RdRp) are indicated.

Fig. 6

Schematic representation of capsid protein (CP) mutants and infectivity assays. (A–I). Rectangles represent open reading frames encoded by CymMV genomic RNA, RNA-dependent RNA polymerase (RdRp), triple gene block ORFs 1, 2, and 3 and capsid protein (CP). Black and gray lines in the CP regions indicate the different amino acids found between pCymMV-M1 and pCymMV-M2, respectively. Position of amino acids is indicated. Chimeric viruses competent in protoplast accumulation and systemic infection in N. benthamiana plants are indicated by +, and the ratio of systemic infected/total inoculated plants is indicated. Systemic infection was detected 2 weeks post-inoculation by RT-PCR. (J) Protoplast infectivity was detected 24 h post-inoculation by northern blot hybridization, and the ribosomal RNA used for a loading control are indicated. Genomic RNA (G), TGBp, and CP subgenomic RNA are indicated. The pCymMV-R- used for a negative control is illustrated in Fig. 1. The pCymMV-R- used as a negative control is illustrated in Fig. 1. The average percentage of relative real-time RT-PCR quantification (from 3 independent experiments) of CymMV RNA from CymMV clone-infected protoplasts at 24 h post-inoculation is indicated below the gels. The accumulation of pCymMV-M1 was set at 100% for relative quantification. Numbers at the left correspond to positions of marker RNAs (sizes in 1000 nucleotides) analyzed in the same gel.

Fig. 7

The amino acid sequence alignment of predicted RNA binding domain of potexvirus CPs and slot-blot detection of biotin-labeled CymMV RNAs. (A) The amino acid sequence alignment of predicted RNA binding domain were conducted by use of clustal X 1.83 (Thompson et al., 1997). The previously identified conserved positive charged amino acids are indicated by stars (Abouhaidar and Lai, 1989). The arrows indicate the amino acids important for pCymMV-M2 to systemically infect N. benthamiana. The viruses, abbreviation and accession number used in alignment are described below. Cymbidium mosaic virus (CymMV, accession number AY571289); Potato aucuba mosaic virus (PAMV, accession number NC_003632; Narcissus mosaic virus (NMV, accession number NC_001441); Scallion virus X (ScaVX, accession number NC_003400); Papaya mosaic virus (PapMV, accession number NC_001748). (B). The concentration of recombinant CPs used in this experiment derived from clones pCymMV-M1 (M1-CP; Fig. 1), -M2 (M2-CP; Fig. 1) and -M2-CP-GL/AP (M2-CP-GL/AP; Fig. 6) and Glutathione-S-transferase (GST) derived from clones (pGEX 2T-1, Pharmacia Biosciences, Inc., New Jersey, USA) are shown. (C) Control experiments using M1 and M2 biotin-labeled RNA only (lane 1 and 2, respectively), or M1 and M2 RNA incubated with GST (lane 3 and 4, respectively) are shown. Because no RNA–protein binding occurred in these control experiments, RNA was detected only in S. (D) The results of experiments using RNA–protein combinations, M1 RNA/M1-CP (lane 1), M2 RNA/M1-CP (lane 2), M1 RNA/M2-CP (lane 3), M2 RNA/M2-CP (lane 4), M1 RNA/M2-CP-GL/AP (lane 5) and M2 RNA/M2-CP-GL/AP (lane 6), are shown. Different concentrations of sodium chloride (in milimolars; mM) added in incubation buffer are indicated. For convenience, the RNA–protein combinations used in the experiments are also indicated in the closed boxes. We repeated this experiment three times and one result is shown. The average S/P ratios were derived from the average of three independent experiments, and data were analyzed by Dunnett's T test. ⁎indicates significant difference (P < 0.01).

Fig. 8

Schematic representation of TGBp1 mutants and infectivity assay between pCymMV-M1 and pCymMV-M2. (A–O) Rectangles represent ORFs encoded by CymMV genomic RNA, RNA-dependent RNA polymerase (RdRp), triple gene block ORFs 1, 2, and 3 and capsid protein (CP). Black and gray lines indicate different amino acids between pCymMV-M1 and pCymMV-M2, respectively. Positions of amino acids are indicated. Chimeric viruses competent in protoplast accumulation and systemic infection in N. benthamiana plants are indicated by +, and the ratio of systemic infected to total inoculated plants is indicated. Systemic infection was detected 2 weeks post-inoculation by RT-PCR. (P) Protoplast infectivity was detected 24 h post-inoculation by northern blot hybridization, and the ribosomal RNA used for a loading control are indicated. Genomic RNA (G), TGBp, and CP subgenomic RNA are indicated. The pCymMV-R- used as a negative control is illustrated in Fig. 1. The average percentage of relative real-time RT-PCR quantification (from 3 independent experiments) of CymMV RNA from CymMV clone-infected protoplasts at 24 h post-inoculation is indicated below the gels. The accumulation of pCymMV-M1 was set at 100% for relative quantification. Numbers at the left correspond to positions of marker RNAs (sizes in 1000 nucleotides) analyzed in the same gel.

Fig. 9

The amino acid sequence alignment of TGBp1 NTP/helicase domain and TGBp3 transmembrane domain of potexvirus. The amino acid sequence alignments were conducted by use of clustal X 1.83 (Thompson et al., 1997). (A) The conserved motifs of the potexvirus NTPase/helicase domain were previously predicted (Kalinina et al., 2002). The 7 predicted NTPase/helicase motifs of potexvirus TGBp1 are shown, and two canonical motifs of NTPase, DEY and GKS, are indicated. The arrows indicate the amino acid positions important for pCymMV-M2 to systemically infect N. benthamiana. (B) The potexvirus TGBp3 transmembrane domain was previously identified (Krishnamurthy et al., 2003). The CymMV transmembrane domain predicted by DAS program (http://www.sbc.su.se/~miklos/DAS/maindas.html) is indicated by a thick black line. The arrows indicate the amino acids important for pCymMV-M2 to systemically infect N. benthamiana. The viruses, abbreviation and accession number used in alignments of TGBp1 NTP/helicase and TGbp3 transmembrane domains are described below. Cymbidium mosaic virus (CymMV, accession number AY571289); Cymbidium mosaic virus (CymMV, accession number AY571289); Foxtail mosaic virus (FMV, accession number NC_001483); Watermelon spotted wilt virus (BSMV, accession number NC_003481); Cassava common mosaic virus (CsCMV, accession number NC_001658); Papaya mosaic virus (PMV, accession number NC_001748); Cactus virus X (CVX, accession number NC_002815); Potato virus X (PVX, accession number NC_001455); White clover mosaic virus (WC1MV, accession number X06728).