JOURNAL OF VIROLOGY, Apr. 2002, p. 3554–3557 0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.7.3554–3557.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 7
Heterologous Movement Protein Strongly Modifies the Infection Phenotype of Cucumber Mosaic Virus Emese Huppert, Dénes Szilassy, Katalin Salánki, Zoltán Divéki, and Ervin Balázs* Agricultural Biotechnology Center, Environmental Biosafety Research Institute, H-2100 Gödöllö, Hungary Received 9 October 2001/Accepted 26 December 2001
A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV. tion of Nicotiana benthamiana and Nicotiana clevelandii plants with in vitro RNA transcripts of pCMV3cymMP, pCMV1, and pCMV2 (19). Northern blot analysis with a radiolabeled probe specific to the complete RNA3 sequence of CMV or to the p22 gene of CymRSV confirmed the presence and identity of the hybrid virus (denoted as CMVcymMP) in both inoculated and noninoculated leaves (Fig. 1B). Total RNA extraction and Northern hybridization were carried out according to standard procedures (16, 20). Electron microscopy showed that CMVcymMP particles isolated from N. benthamiana by a standard CMV isolation procedure (8) were indistinguishable from CMV particles (data not shown). The yield of CMVcymMP purification was comparable to that of CMV. Several plant species were inoculated with CMVcymMP virions to examine the host range and infection phenotype of the hybrid virus. Parallel infection was carried out with CMV and CymRSV for comparison. The infection was evaluated by visual observation and Northern blot analysis (Table 1). The hybrid virus established local infection on Chenopodium quinoa, Chenopodium foetidum, and Chenopodium murale 5 to 7 days postinoculation (dpi). In these hosts, the size and morphology of local lesions induced by CMVcymMP were clearly determined by the foreign MP gene. This is in agreement with the observation that the MPs play a central role in determination of local lesion phenotype (10, 18). CMVcymMP systemically infected all the tested systemic hosts common to CMV and CymRSV. The small, scattered bleached spots on N. benthamiana, small necrotic spots on N. clevelandii, and leaf distortion on Nicotiana megalosiphon plants indicated the decisive effect of the CymRSV MP on the symptom phenotype. Unlike CymRSV, the hybrid virus caused systemic infection of Nicotiana debneyi and N. tabacum cv. Xanthi, indicating the ability of CymRSV MP and other CMV proteins and host factors to establish proper interactions for systemic movement. In contrast to CMV, both CMVcymMP and CymRSV caused local lesions on Nicotiana glutinosa. Similar observations were reported by Scholthof and colleagues (17), who analyzed the
Plant viruses exploit and modify the preexisting endogenous pathways for macromolecular movement during their spread in the host plant, which requires successful interactions between the movement protein (MP) and other viral and host components (4, 9). Cymbidium ringspot tombusvirus (CymRSV) and cucumber mosaic cucumovirus (CMV) are distantly related singlestranded RNA viruses. The genome of CMV consists of five genes expressed from three genomic and two subgenomic RNAs (7, 11). The 5⬘ region of RNA3 codes for the MP, and the coat protein (CP) gene is located on the 3⬘ end of the same RNA. The monopartite CymRSV genome contains five ORFs (13). The MP (p22) is encoded by ORF4, which is nested with ORF5. The latter encodes the p19 protein, which aggravates the symptoms (17). These two viruses also differ in their requirements for movement in the infected plants. Although both viruses are thought to move in the form of nucleoprotein complexes in the nonvascular tissue (1, 12), CMV has been shown to require MP as well as CP for this process (2, 3), while CP-defective tombusviruses were able to move between the neighboring cells and even systemically in some Nicotiana species (6). In this study, we provide further evidence for the interchangeable nature of viral MPs, between unrelated species, and for the role of CymRSV MP in symptom formation. Symptom induction and replication of CMVcymMP. A hybrid Trk7-CMV RNA3 was used to examine whether the MP of CymRSV could substitute for the MP of CMV. The MP gene of the cDNA clone pCMV3 (Fig. 1A) (15) was replaced by the CymRSV p22 gene employing standard PCR and cloning techniques, resulting in pCMV3cymMP (Fig. 1A). The p22 gene was amplified from Cym19stop clone of CymRSV (courtesy of J. Burgyán), with primers C22–5⬘ (5⬘-GCACTAGTCA TGGACACTGAATACCAAC-3⬘), and C22–3⬘ (5⬘-GCACCG GTCTAGACTGAAGAGTCTGTCC-3⬘). The conformity of the foreign MP gene was verified by sequencing. Infectivity of the construct was tested by coinocula* Corresponding author. Mailing address: Agricultural Biotechnology Center, Environmental Biosafety Research Institute, H-2101 Gödöllö, Hungary. Phone: 36 28 430 539. Fax: 36 28 430 150. E-mail:
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FIG. 1. (A) Schematic representation of the various CMV RNA3 constructs used in this study. pCMV3 is the cDNA clone of the wildtype Trk7-CMV RNA3. MP and CP genes are indicated by left and right black rectangles, respectively. In pCMV3cymMP, the CMV MP gene has been replaced with the MP gene of CymRSV, indicated by a light gray rectangle. In pCMV3⌬CP and pCMV3cymMP⌬CP, a 200nucleotide deletion was created in the 3⬘-proximal end of the CMV CP gene. The deleted region is indicated by the empty rectangle. In pCMV3⌬CP-GFP and pCMV3cymMP⌬CP-GFP, the CMV CP gene was replaced by a GFP gene (gray rectangle). In vitro RNA transcripts of each construct were coinoculated with cDNA-derived transcripts of Trk7-CMV RNAs 1 and 2, to give CMV, CMVcymMP, CMV⌬CP, CMVcymMP⌬CP, CMV⌬CP-GFP, and CMVcymMP⌬CP-GFP, respectively. (B) Representative Northern blot analysis of viral RNAs isolated from inoculated (I) and systemic (S) leaves of N. benthamiana infected with CMV or CMVcymMP as indicated. RNAs from an equal area of mock-inoculated N. benthamiana leaf are presented in lane M. The radiolabeled probes were specific to CMV RNA3 (I) or the CymRSV MP gene (II). The positions of the CMV RNAs are indicated. (C) Northern blot analysis of total RNAs extracted from inoculated leaves of N. benthamiana plants infected with CMV (lane 1), CMV⌬CP (lane 2), CMVcymMP (lane 3), or CMVcymMP⌬CP (lane 4). The radiolabeled probe was specific to CMV RNA3. The exposure times were 4 h for CMV, CMV⌬CP, and CMVcymMP and 24 h for CMVcymMP⌬CP. The positions of the viral RNAs are indicated.
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influence of the MP gene of tomato bushy stunt tombusvirus (TBSV) on disease symptoms. As estimated from the intensity of bands in Northern blots, the amounts of viral RNA3 in leaves infected with either of the three viruses were comparable, except for the systemic leaves of N. debneyi and N. tabacum cv. Xanthi, in which the amount of CMVcymMP RNA3 was significantly less than that of CMV (data not shown). Effects of deletion in the CP gene. To determine whether the CymRSV MP can promote cell-to-cell movement of CMV in the absence of CMV CP, pCMV3cymMP⌬CP (Fig. 1A) was generated by eliminating the region between the ApaI and Tth111I restriction sites in the CP gene. An analogous deletion mutant, pCMV3⌬CP (Fig. 1A), was used as a control. Systemic hosts of both CMV and CMVcymMP (N. benthamiana, N. clevelandii, N. megalosiphon, N. debneyi, and N. tabacum cv. Xanthi) were infected with pCMV3, pCMVcymMP, pCMV3⌬CP, and pCMVcymMP⌬CP RNA transcripts in the presence of pCMV1 and pCMV2 transcripts. In plants infected with CMV and CMVcymMP, systemic symptoms appeared 7 to 10 dpi. In the case of CMV⌬CP-inoculated plants, no visible sign of virus infection could be observed, and no viral RNA could be detected in the inoculated leaf by Northern blot analysis, which is consistent with previous reports (2, 3). CMVcymMP⌬CP-inoculated plants did not show local or systemic symptoms, but Northern analysis of the inoculated leaves indicated the presence of viral RNAs (Fig. 1C). However, the level of CMVcymMP⌬CP RNA3 was significantly lower than those of CMV and CMVcymMP. No CMVcymMP⌬CP RNA3 was detected in the systemic leaves of the inoculated plants (data not shown). In order to monitor the spread of CP-deficient constructs, a green fluorescent protein (GFP) gene was inserted into their genomes, to give CMVcymMP⌬CP-GFP and CMV⌬CP-GFP (Fig. 1A). The GFP gene was amplified from clone TU#65 (5) with oligonucleotides M13-28 and 5⬘ GFP/NdeI (5⬘-CGCA TATGAGTAAAGGAG-3⬘). Plants were illuminated with a 100-W long-wavelength UV lamp and photographed with Kodak Elite ChromeII film and a Canon Y1 filter. For epifluorescence microscopy, leaves were viewed with an Olympus IMT-2 microscope with filter set BP405/Y455. In C. quinoa inoculated with CMV⌬CP-GFP, no signs of infection were visible under UV illumination, but epifluorescence microscopy revealed numerous isolated infected cells. Similar results were observed on inoculated systemic hosts of CMV. Thus, infection by CMV⌬CP-GFP was restricted to single inoculated cells (Fig. 2A). CMVcymMP⌬CP-GFP caused development of fluorescent foci in C. quinoa, which were clearly visible under UV illumination. Epifluorescence microscopy proved that GFP expression was not confined to the initially infected cells (Fig. 2B). In N. benthamiana and N. clevelandii plants inoculated with CMVcymMP⌬CP-GFP, fluorescent patches were observed in the inoculated leaves 4 to 10 dpi (Fig. 2C and D). Similar fluorescence was found in other systemic hosts common to CMV and CMVcymMP. Fluorescence could not be detected in the noninoculated leaves of these plants, suggesting the absence of long-distance movement of CMVcymMP⌬CP-GFP. These observations show that the MP of CymRSV has a
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FIG. 2. Development of fluorescence in plants infected with GFP-expressing derivatives of CMV. (A) Single epidermal cells of C. quinoa infected with CMV⌬CP-GFP (4 dpi). Fluorescent cells are indicated by arrows. (B) Cluster of C. quinoa cells infected with CMVcymMP⌬CP-GFP (4 dpi). (C and D) N. benthamiana (C) and N. clevelandii (D) plants infected with CMVcymMP⌬CP-GFP showing fluorescent foci in inoculated leaves 7 dpi. Inoculated leaves are indicated (I). Scale bar, 20 m.
broader capability to promote virus movement than does the CMV MP. Similar features of the groundnut rosette umbravirus (GRV) MP were demonstrated in an analogous experimental system (14). Like the hybrid CMV construct expressing GRV MP, the CymRSV MP together with the CMV CP enabled the virus to enter and spread through the vascular system in certain hosts. Constructs expressing the CymRSV MP without functional CMV CP (CMVcymMP⌬CP and CMVcymMP⌬CP-GFP) spread efficiently from cell to cell, but were confined to the inoculated nonvascular tissue. It can be concluded that although p22 can facilitate CymRSV nucleoprotein to cross the bundle sheath-phloem interface
without the CymRSV CP, it requires the presence of CMV CP to promote entry and spread of the hybrid CMV through the vascular system. Since CMV is proposed to move systemically in the form of virus particles (1), the inability of CP mutant constructs to form virions could explain the lack of long-distance movement function. This work was supported by grants from the VRTP-Impact EU Project (5th Framework Program) and the Hungarian OTKA (032737). K.S. was the recipient of a Bólyai scholarship (Hungarian Academy of Sciences). We also thank Mark Tepfer for editorial help in preparing the manuscript.
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TABLE 1. Assay of infectivity of parental and chimeric viruses Infectivity assay resultb CMV
Hosta I
S
CymRSV RNA I
S
I
S
CMVcymMP RNA I
S
I
S
RNA I
S
Group I C. quinoa C. foetidum C. murale
NL 1 CL 1 NL
NI NI NI
⫹ ⫹ ⫹
⫺ ⫺ ⫺
s NL NL vs NL
NI NI NI
⫹ ⫹ ⫹
⫺ ⫺ ⫺
s NL NL vs NL
NI NI NI
⫹ ⫹ ⫹
⫺ ⫺ ⫺
Group II N. benthamiana N. clevelandii N. megalosiphon
M M sev N
M, D M, D sev N
⫹ ⫹ ⫹
⫹ ⫹ ⫹
M NL, CL 1 CL, D
sev M, D sev M, D Mild M, D
⫹ ⫹ ⫹
⫹ ⫹ ⫹
CL, M CL 1 CL
sev M, D sev M, D Mild M, D
⫹ ⫹ ⫹
⫹ ⫹ ⫹
Group III N. debneyi N. tabacum cv. Xanthi N. glutinosa
Mild M M M
Mild M, D M M
⫹ ⫹ ⫹
⫹ ⫹ ⫹
NS NL NL
NI NI NI
⫹ ⫹ ⫹
⫺ ⫺ ⫺
NS Mild M NL, CL
Mild M Mild M NI
⫹ ⫹ ⫹
⫹ ⫹ ⫺
a
Host group I, common local lesion hosts of CMV and CymRSV; host group II, systemic hosts of both CMV and CymRSV; host group III, other hosts examined. b I, inoculated leaves; S, systemic leaves; NL, necrotic lesion; CL, chlorotic lesion; M, mosaic; D, leaf distortion; N, necrosis; NS, no symptoms; NI, noninfected; l, large; s, small; vs, very small; sev, severe. CMV and CymRSV RNA3s, respectively, were detected by Northern analysis. ⫹, presence of RNAs; ⫺, absence of RNAs.
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